Method of producing L-lysine by fermentation

ABSTRACT

A bacterium belonging to the genus Escherichia, which is transformed by introducing, into its cells, a DNA coding for a dihydrodipicolinate synthase originating from a bacterium belonging to the genus Escherichia having mutation to desensitize feedback inhibition by L-lysine and a DNA coding for an aspartokinase III originating from a bacterium belonging to the genus Escherichia having mutation to desensitize feedback inhibition by L-lysine; preferably a bacterium belonging to the genus Escherichia in which a dihydrodipicolinate reductase gene and a diaminopimelate dehydrogenase gene originating from Brevibacterium lactofermentum (or a succinyldiaminopimelate transaminase gene and a succinyldiaminopimelate deacylase gene) are further enhanced, is cultivated in an appropriate medium, L-lysine is produced and accumulated in a culture thereof, and L-lysine is collected from the culture.

TECHNICAL FIELD

The present invention relates to microbial industry, and in particularrelates to a method of producing L-lysine by fermentation, DNA's andmicroorganisms to be used for this production method.

BACKGROUND ART

In the prior art, when L-lysine is produced by a fermentative method, amicrobial strain separated from the natural environment or an artificialmutant strain obtained from such a microbial strain is used in order toimprove the productivity. A large number of artificial mutant strainsproducing L-lysine are known. Most of them are S-2-aminoethylcysteine(AEC) resistant mutant strains, and belong to the genus ofBrevibacterium, Corynebacterium, Bacillus or Escherichia. Further,various techniques have been disclosed for increasing amino acidproduction, for example, by employing a transformant using recombinantDNA (U.S. Pat. No. 4,278,765).

With respect to those belonging to the genus Escherichia, for example,Japanese Patent Application Laid-open No. 56-18596, U.S. Pat. No.4,346,170, and Applied Microbiology and Biotechnoloay, 15, 227-231(1982) describe methods of producing L-lysine using a bacterial strainin which dihydrodipicolinate synthase (hereinafter sometimes abbreviatedas "DDPS") is enhanced. However, DDPS used in these cases is a wildtype, which suffers feedback inhibition by L-lysine. Thus sufficientlysatisfactory L-lysine productivity has not been obtained. Incidentally,Applied Microbiology and Biotechnology, 15, 227-231 (1982) mentionedabove is describes an L-lysine production of 3 g/l of L-lysinehydrochloride from 75 g/l of glucose, wherein a consumption coefficient(number of g of L-lysine produced from 1 g of sugar, or percentagethereof) is calculated to be 0.04, or 4%.

On the other hand, Korean Patent Publication No. 92-8382 describes amethod of producing L-lysine using a bacterium belonging to Escherichiato which DDPS originating from a bacterium belonging to the genusCorynebacterium, which is known not to suffer feedback inhibition byL-lysine (consumption coefficient: 17%), is introduced. However, theupper limit temperature for growth of bacteria belonging to the genusCorynebacterium is lower than the upper limit temperature for growth ofbacteria belonging to the genus Escherichia by about 10 degrees. Thus itseems that cultivation should be performed at a lowered cultivationtemperature if DNA coding for DDPS originating from a bacteriumbelonging to the genus Corynebacterium is introduced into a bacteriumbelonging to the genus Escherichia in order to utilize it for L-lysineproduction. Therefore, it is anticipated that it is difficult to exhibitadvantages possessed by the bacterium belonging to the genus Escherichiathat the growth temperature is high, the growth speed is fast, and theL-lysine-producing speed is also fast. Generally, when a geneoriginating from a heterologous organism is expressed, there areoccasionally caused decomposition of an expression product by proteaseand formation of an insoluble inclusion body, in which more difficultiesare anticipated as compared with a case of expression of a homologousgene. Further, when DNA coding for DDPS originating from a bacteriumbelonging to the genus Corynebacterium is introduced into a bacteriumbelonging to the genus Escherichia to industrially produce L-lysine,more strict regulation is obliged as compared with a case of use of arecombinant to which a homologous gene is introduced, in accordance withthe recombinant DNA guideline.

By the way, the dihydrodipicolinate synthase (DDPS) is an enzyme fordehydrating and condensing aspartosemialdehyde and pyruvic acid tosynthesize dihydrodipicolinic acid. This reaction is located at anentrance into a branch to proceed to an L-lysine biosynthesis system inbiosynthesis of amino acids of the aspartic acid family. This enzyme isknown to be in charge of an important regulatory site as aspartokinaseis in bacteria belonging to the genus Escherichia.

DDPS is encoded by a gene called dapA in E. coli (Escherichia coli). ThedapA has been cloned, and its nucleotide sequence has been alsodetermined (Richaud, F. et al., J. Bacteriol., 297 (1986)).

On the other hand, aspartokinase (hereinafter sometimes abbreviated as"AK") is an enzyme for catalyzing a reaction to convert aspartic acidinto β-phosphoaspartic acid, which serves as a main regulatory enzyme ina biosynthesis system of amino acids of the aspartic acid family. AK ofE. coli has three types (AKI, AKII, AKIII), two of which are complexenzymes with homoserine dehydrogenase (hereinafter sometimes abbreviatedas "HD"). One of the complex enzymes is AKI-HDI encoded by a thrA gene,and the other is AKII-HDII encoded by a metLM gene. AKI is subjected toconcerted suppression by threonine and isoleucine and inhibited bythreonine, while AKII is suppressed by methionine.

On the contrary, it is known that only AKIII is a simple functionenzyme, which is a product of a gene designated as lysC, and issubjected to suppression and feedback inhibition by L-lysine. The ratioof their intracellular activities is AKI:AKII:AKIII=about 5:1:4.

As described above, DDPS originating from bacteria belonging to thegenus Corynebacterium is not subjected to feedback inhibition byL-lysine. However, when it is introduced into a bacterium belonging tothe genus Escherichia to utilize it for L-lysine production, a problemarises in the cultivation temperature. It is expected that L-lysine canbe efficiently produced by fermentation by using a bacterium belongingto the genus Escherichia if a mutant enzyme of DDPS or AKIII originatingfrom a bacterium belonging to the genus Escherichia, which is notsubjected to feedback inhibition by L-lysine, can be obtained. However,there is no preceding literature which describes such a mutant enzyme ofDDPS, and although there is one report on a mutant enzyme of AKIII (Boy,E., et al., J. Bacteriol., 112, 84 (1972)) no example has been knownwhich suggests that such a mutant enzyme may improve productivity ofL-lysine.

DISCLOSURE OF THE INVENTION

The present invention has been made taking the aforementioned viewpointsinto consideration, an object of which is to obtain DDPS and AKIIIoriginating from bacteria belonging to the genus Escherichia withsufficiently desensitized feedback inhibition by L-lysine, and provide amethod of producing L-lysine by fermentation which is more improved thanthose in the prior art.

As a result of diligent and repeated investigation in order to achievethe object described above, the present inventors have succeeded inobtaining DNA coding for DDPS originating from a bacterium belonging tothe genus Escherichia in which feedback inhibition by L-lysine issufficiently desensitized. The DNA coding for DDPS originating from E.coli in which feedback inhibition by L-lysine is sufficientlydesensitized is sometimes referred to herein as mutant dapA or dapA*.

The inventors have further created a bacterium belonging to the genusEscherichia harboring mutant dapA and aspartokinase which isdesensitized feedback inhibition by L-lysine. The DNA coding foraspartokinase originating from E. coli in which feedback inhibition byL-lysine is sufficiently desensitized is sometimes referred to herein asmutant lysC or lysC*.

The inventors have further created a bacterium belonging to the genusEscherichia harboring mutant dapA and mutant lysC. And it has been foundthat a considerable amount of L-lysine can be produced and accumulatedin a culture by cultivating the aforementioned bacterium belonging tothe genus Escherichia in a preferred medium.

The inventors have still further found that the productivity of L-lysinecan be further improved by enhancing other genes in the L-lysinebiosynthesis system of a bacterium belonging to the genus Escherichiaharboring the mutant dapA and the mutant lysC.

Namely, the present invention lies in a DNA coding for adihydrodipicolinate synthase originating from a bacterium belonging tothe genus Escherichia having mutation to desensitize feedback inhibitionby L-lysine. The mutation to desensitize feedback inhibition by L-lysineis exemplified by mutation selected from the group consisting ofmutation to replace a 81st alanine residue with a valine residue,mutation to replace a 118th histidine residue with a tyrosine residue,and mutation to replace the 81st alanine residue with the valine residueand replace the 118th histidine residue with the tyrosine residue, ascounted from the N-terminal in an amino acid sequence ofdihydrodipicolinate synthase defined in SEQ ID NO:4 in Sequence Listing.

The present invention further lies in a bacterium belonging to the genusEscherichia transformed by introducing, into its cells, a DNA coding fora dihydrodipicolinate synthase originating from a bacterium belonging tothe genus Escherichia having mutation to desensitize feedback inhibitionby L-lysine. The mutation to desensitize feedback inhibition by L-lysineis exemplified by mutation to replace a 81st alanine residue with avaline residue, mutation to replace a 118th histidine residue with atyrosine residue, and mutation to replace the 81st alanine residue withthe valine residue and replace the 118th histidine residue with thetyrosine residue, as counted from the N-terminal in an amino acidsequence of dihydrodipicolinate synthase defined in SEQ ID NO:4 inSequence Listing.

The present invention further lies in the aforementioned bacteriumbelonging to the genus Escherichia harboring an aspartokinase which isalso desensitized feedback inhibition by L-lysine. A method to allow thebacterium belonging to the genus Escherichia to harbor the aspartokinasewhich is desensitized feedback inhibition by L-lysine is exemplified bya method for introducing, into its cells, a DNA coding for anaspartokinase III originating from a bacterium belonging to the genusEscherichia having mutation to desensitize feedback inhibition byL-lysine.

The mutation of the aspartokinase III to desensitize feedback inhibitionby L-lysine is exemplified by mutation to replace a 323rd glycineresidue with an aspartic acid residue, mutation to replace the 323rdglycine residue with the aspartic acid residue and replace a 408thglycine residue with an aspartic acid residue, mutation to replace a34th arginine residue with a cysteine residue and replace the 323rdglycine residue with the aspartic acid residue, mutation to replace a325th leucine residue with a phenylalanine residue, mutation to replacea 318th methionine residue with an isoleucine residue, mutation toreplace the 318th methionine residue with the isoleucine residue andreplace a 349th valine residue with a methionine residue, mutation toreplace a 345th serine residue with a leucine residue, mutation toreplace a 347th valine residue with a methionine residue, mutation toreplace a 352nd threonine residue with an isoleucine residue, mutationto replace the 352nd threonine residue with the isoleucine residue andreplace a 369th serine residue with a phenylalanine residue, mutation toreplace a 164th glutamic acid residue with a lysine residue, andmutation to replace a 417th methionine residue with an isoleucineresidue and replace a 419th cysteine residue with a tyrosine residue, ascounted from the N-terminal in an amino acid sequence of aspartokinaseIII defined in SEQ ID NO:8 in Sequence Listing.

The DNA coding for a dihydrodipicolinate synthase originating from abacterium belonging to the genus Escherichia having mutation todesensitize feedback inhibition by L-lysine, and the DNA coding for anaspartokinase III having mutation to desensitize feedback inhibition byL-lysine may be harbored on a chromosome of a bacterium belonging to thegenus Escherichia respectively, or may be harbored in cells on anidentical plasmid or separate plasmids. Further, it is also acceptablethat one of the respective DNA's is harbored on a chromosome, and theother DNA is harbored on a plasmid.

The present invention still further lies in the aforementioned bacteriumbelonging to the genus Escherichia wherein a dihydrodipicolinatereductase gene is enhanced. The enhancement of the dihydrodipicolinatereductase gene can be achieved by transformation with a recombinant DNAconstructed by ligating the dihydrodipicolinate reductase gene with avector autonomously replicable in cells of bacteria belonging to thegenus Escherichia.

The present invention further lies in the aforementioned bacteriumbelonging to the genus Escherichia wherein an enhanced diaminopimelatedehydrogenase gene originating from coryneform bacteria such asBrevibacterium lactofermentum is introduced. The introduction of theenhanced diaminopimelate dehydrogenase gene originating from coryneformbacteria can be achieved by transformation with a recombinant DNAconstructed by ligating the gene with a vector autonomously replicablein cells of bacteria belonging to the genus Escherichia. As coryneformbacteria, there may be exemplified wild type strains producing glutamicacid, and mutant strains thereof producing other amino acids, whichbelong to the genus Corynebacterium or the genus Brevibacterium. Moreconcretely, Brevibacterium flavum, Brevibacterium divaricatum,Corynebacterium glutamicum and Corynebacterium lilium as well asBrevibacterium lactofermentum are exemplified as coryneform bacteriaused for the present invention.

The present invention further lies in the bacterium belonging to thegenus Escherichia wherein a tetrahydrodipicotinate succinylase gene anda succinyldiaminopmelate deacylase gene are enhanced instead of theaforementioned diaminodipimelate dehydrogenase gene. The enhancement ofthese genes can be achieved by transformation with a single recombinantDNA or two recombinant DNA's constructed by ligating these genes with anidentical vector or different vectors autonomously replicable in cellsof bacteria belonging to the genus Escherichia.

The present invention further provides a method of producing L-lysinecomprising the steps of cultivating any of the bacteria belonging to thegenus Escherichia described above in an appropriate medium, producingand accumulating L-lysine in a culture thereof, and collecting L-lysinefrom the culture.

In this specification, DNA coding for DDPS or AKIII, or DNA containing apromoter in addition thereto is sometimes referred to as "DDPS gene" or"AKIII gene". Further, the mutant enzyme which is desensitized feedbackinhibition by L-lysine, and DNA coding for it or DNA containing apromoter in addition to it are sometimes simply refereed to as "mutantenzyme" and "mutant gene", respectively. Further, the phrase "feedbackinhibition by L-lysine is desensitized" means that substantialdesensitization of inhibition is sufficient, and completedesensitization is not necessary.

The present invention will be explained in detail below.

<1> DNA Coding for Mutant Dihydrodipicolinate Synthase (DDPS) of thePresent Invention

The DNA coding for the mutant DDPS of the present invention has mutationto desensitize feedback inhibition by L-lysine of DDPS encoded in DNAcoding for the wild type DDPS. DDPS is exemplified by those originatingfrom bacteria belonging to the genus Escherichia, especially DDPSoriginating from E. coli The mutation of DDPS to desensitize feedbackinhibition by L-lysine is exemplified by:

(1) mutation to replace a 81st alanine residue with a valine residue;

(2) mutation to replace a 118th histidine residue with a tyrosineresidue; and

(3) mutation to replace the 81st alanine residue with the valine residueand replace the 118th histidine residue with the tyrosine residue; ascounted from the N-terminal of DDPS in an amino acid sequence of DDPSdefined in SEQ ID NO:4 in Sequence Listing.

The DNA coding for the wild type DDPS is not especially limited providedthat it codes for DDPS originating from a bacterium belonging to thegenus Escherichia, which is concretely exemplified by DNA coding for anamino acid sequence defined in SEQ ID NO:4, and is further concretelyexemplified by a sequence represented by base numbers 272-1147 in a basesequence defined in SEQ ID NO:3. In these sequences, those having themutation in nucleotide sequence to cause the replacement of amino acidresidues described above are the DNA coding for the mutant DDPS of thepresent invention. Any codon corresponding to the replaced amino acidresidue is available especially irrelevantly to its kind, provided thatit codes for the identical amino acid residue. Further, it is postulatedthat possessed DDPS is slightly different in sequence depending ondifference in bacterial species and bacterial strain, however, thosehaving replacement, deletion or insertion of amino acid residue(s) atposition(s) irrelevant to enzyme activity are also included in themutant DDPS gene of the present invention.

A method for obtaining such a mutant gene is as follows. At first, a DNAcontaining a wild type DDPS gene or DDPS gene having another mutation issubjected to an in vitro mutation treatment, and a DNA after themutation treatment is ligated with a vector DNA adapted to a host toobtain a recombinant DNA. The recombinant DNA is introduced into a hostmicroorganism to obtain transformants. When one which expresses a mutantDDPS is selected among the aforementioned transformants, such atransformant harbors a mutant gene. Alternatively, a DNA containing awild type DDPS gene or DDPS gene having another mutation may be ligatedwith a vector DNA adapted to a host to obtain a recombinant DNA. Therecombinant DNA is thereafter subjected to an in vitro mutationtreatment, and a recombinant DNA after the mutation treatment isintroduced into a host microorganism to obtain transformants. When onewhich expresses a mutant DDPS is selected among the aforementionedtransformants, such a transformant also harbors a mutant gene.

It is also acceptable that a microorganism which, produces a wild typeenzyme is subjected to a mutation treatment to create a mutant strainwhich produces a mutant enzyme, and then a mutant gene is obtained fromthe mutant strain. Alternatively, a transformant to which a recombinantDNA ligated with a wild type gene is introduced may be subjected to amutation treatment to create a mutant strain which produces a mutantenzyme. When a recombinant DNA is thereafter recovered from the mutantstrain, a mutant gene is created on the aforementioned DNA.

The agent for performing the in vitro mutation treatment of DNA isexemplified by hydroxylamine and the like. Hydroxylamine is a chemicalmutation treatment agent which causes mutation from cytosine to thymineby changing cytosine to N⁴ -hydroxycytosine. Alternatively, when amicroorganism itself is subjected to a mutation treatment, the treatmentis performed by using ultraviolet light irradiation, or a mutating agentusually used for artificial mutation such asN-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid.

No problem occurs when any one is used as a donor microorganism for DNAcontaining the wild type DDPS gene or DDPS gene having another mutationdescribed above, provided that it is a microorganism belonging to thegenus Escherichia. Concretely, it is possible to utilize those describedin a book written by Neidhardt et al. (Neidhardt, F. C. et al.,Escherichia coli and Salmonella Typhimurium, American Society forMicrobiology, Washington D. C., 1208, table 1). For example, an E. coliJM109 strain and an MC1061 strain are exemplified. When a wild strain isused as a donor microorganism for DNA containing a DDPS gene, a DNAcontaining a wild type DDPS gene can be obtained.

(1) Preparation of Wild Type DDPS Gene

An example of preparation of DNA containing a DDPS gene will bedescribed below. At first, E. coli having wild type dapA, for example,MC1061 strain, is cultivated to obtain a culture. When the microorganismdescribed above is cultivated, cultivation may be performed inaccordance with an ordinary solid culture method, however, cultivationis preferably performed by adopting a liquid culture method consideringefficiency during collection of the bacterium. A medium may be used inwhich one or more nitrogen sources such as yeast extract, peptone, meatextract, corn steep liquor and exudate of soybean or wheat are addedwith one or more inorganic salts such as potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride,magnesium chloride, ferric chloride, ferric sulfate or manganesesulfate, and further optionally and adequately added with sugarmaterials, vitamins and the like. It is appropriate that the initial pHof the medium is adjusted to 6-8. The cultivation is performed for 4-24hours at 30-42° C., preferably at about 37° C. by means of deep culturewith aeration and agitation, shaking culture or stationary culture orthe like.

The culture thus obtained is centrifuged, for example, at 3,000 r.p.m.for 5 minutes to obtain a cell pellet of E. coli MC1061 strain.Chromosomal DNA can be obtained from the cell pellet by means of, forexample, a method of Saito and Miura (Biochem. Biophys. Acta., 72, 619(1963)), or a method of K. S. Kirby (Biochem. J., 64, 405 (1956)).

In order to isolate the DDPS gene from the chromosomal DNA thusobtained, a chromosomal DNA library is prepared. At first, thechromosomal DNA is partially digested with a suitable restriction enzymeto obtain a mixture of various fragments. A wide variety of restrictionenzymes can be used if the degree of cutting is controlled by thecutting reaction time and the like. For example, Sau3AI is allowed toreact on the chromosomal DNA at a temperature not less than 30° C.,preferably at 37° C. at an enzyme concentration of 1-10 units/ml forvarious periods of time (1 minute to 2 hours) to digest it.

Next, obtained DNA fragments are ligated with a vector DNA autonomouslyreplicable in cells of bacteria belonging to the genus Escherichia toprepare recombinant DNA. Concretely, a restriction enzyme, whichgenerates the terminal nucleotide sequence complement to that generatedby the restriction enzyme Sau3AI used to cut the chromosomal DNA, forexample, BmHI, is allowed to act on the vector DNA under a condition ofa temperature not less than 30° C. and an enzyme concentration of 1-100units/ml for not less than 1 hour, preferably for 1-3 hours tocompletely digest it, and cut and cleave it. Next, the chromosomal DNAfragment mixture obtained as described above is mixed with the cleavedand cut vector DNA, on which DNA ligase, preferably T4 DNA ligase isallowed to act under a condition of a temperature of 4-16° C. at anenzyme concentration of 1-100 units/ml for not less than 1 hour,preferably for 6-24 hours to obtain recombinant DNA.

The obtained recombinant DNA is used to transform a microorganismbelonging to the genus Escherichia, for example, a DDPS deficient mutantstrain such as an Escherichia coli K-12 strain, preferably a JE7627strain (ponB704, dacB12, pfv⁺, tonA2, dapA, lysA, str, malA38, metB1,ilvH611, leuA371, proA3, lac-3, tsx-76) to prepare a chromosomal DNAlibrary. The transformation can be performed, for example, by a methodof D. M. Morrison (Methods in Enzymology 68, 326 (1979)) or a method inwhich recipient bacterial cells are treated with calcium chloride toincrease permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol.,53, 159 (1970)). The JE7627 strain is available from National Instituteof Genetics (Mishima-shi, Shizuoka-ken, Japan).

A bacterial strain having recombinant DNA of the DDPS gene is obtainedfrom strains having increased DDPS activity or strains in whichauxotrophy resulting from deficiency in DDPS gene is complemented, amongthe obtained chromosomal DNA library. For example, a DDPS deficientmutant strain requires diaminopimelic acid. Thus when the DDPS deficientmutant strain is used as a host, a DNA fragment containing the DDPS genecan be obtained by isolating a bacterial strain which becomes capable ofgrowing on a medium containing no diaminopimelic acid, and recoveringrecombinant DNA from the bacterial strain.

Confirmation of the fact whether or not a candidate strain havingrecombinant DNA containing a DDPS gene actually harbors recombinant DNAin which the DDPS gene is cloned can be achieved by preparing a cellularextract from the candidate strain, and preparing a crude enzyme solutiontherefrom to confirm whether or not the DDPS activity has beenincreased. A procedure to measure the enzyme activity of DDPS can beperformed by a method of Yugari et al. (Yugari, Y. and Gilvarg, C., J.Biol. Chem., 240, 4710 (1962)).

Recombinant DNA in which DNA containing the DDPS gene is inserted intothe vector DNA can be isolated from the bacterial strain described aboveby means of, for example, a method of P. Guerry et al. (J. Bacteriol.,116, 1064 (1973)) or a method of D. B. Clewell (J. Bacteriol., 110, 667(1972)).

Preparation of the wild type DDPS gene can be also performed bypreparing chromosomal DNA from a strain having a DDPS gene on chromosomeby means of a method of Saito and Miura or the like, and amplifying theDDPS gene by means of a polymerase chain reaction (PCR) method (seeWhite, T. J. et al.; Trends Genet., 5, 185 (1989)). DNA primers to beused for the amplification reaction are those complemental to both3'-terminals of a double stranded DNA containing an entire region or apartial region of the DDPS gene. When only a partial region of the DDPSgene is amplified, it is necessary to use such DNA fragments as primersto perform screening of a DNA fragment containing the entire region froma chromosomal DNA library. When the entire region of the DDPS gene isamplified, a PCR reaction solution including DNA fragments containingthe amplified DDPS gene is subjected to agarose gel electrophoresis, andthen an aimed DNA fragment is extracted. Thus a DNA fragment containingthe DDPS gene can be recovered.

The DNA primers may be adequately prepared on the basis of, for example,a sequence known in E. coli (Richaud, F. et al., J. Bacteriol., 297(1986)). Concretely, primers which can amplify a region comprising 1150bases coding for the DDPS gene are preferable, and two species ofprimers defined in SEQ ID NO:1 and NO:2 are suitable. Synthesis of theprimers can be performed by an ordinary method such as a phosphoamiditemethod (see Tetrahedron Letters, 22, 1859 (1981)) by using acommercially available DNA synthesizer (for example, DNA SynthesizerModel 380B produced by Applied Biosystems Inc.). Further, the PCR can beperformed by using a commercially available PCR apparatus (for example,DNA Thermal Cycler Model PJ2000 produced by Takara Shuzo Co., Ltd.),using Tag DNA polymerase (supplied by Takara Shuzo Co., Ltd.) inaccordance with a method designated by the supplier.

With respect to the DDPS gene amplified by the PCR method, operationssuch as introduction of mutation into the DDPS gene become easy, when itis ligated with a vector DNA autonomously replicable in cells ofbacteria belonging to the genus Escherichia, and introduced into cellsof bacteria belonging to the genus Escherichia. The vector DNA to beused, the transformation method, and the confirmation method for thepresence of the DDPS gene are the same as those in the aforementionedprocedure.

(2) Introduction of Mutation Into DDPS Gene

The method for carrying out mutation such as replacement, insertion anddeletion of amino acid residues is exemplified by a recombinant PCRmethod (Higuchi, R., 61, in PCR Technology (Erlich, H. A. Eds., Stocktonpress (1989))), and a site specific mutagenesis method (Kramer, W. andFrits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel T. A. et al.,Meth. in Enzymol., 154, 367 (1987)). Aimed mutation can be caused at anaimed site by using these methods.

Further, according to chemical synthesis of an aimed gene, it ispossible to introduce mutation or random mutation into an aimed site.

Further, a method is available in which the DDPS gene on chromosome orplasmid is directly treated with hydroxylamine (Hashimoto, T. andSekiguchi, M. J. Bacteriol., 159, 1039 (1984)). Alternatively, it isacceptable to use a method in which a bacterium belonging to the genusEscherichia having the DDPS gene is irradiated by ultraviolet light, ora method based on a treatment with a chemical agent such asN-methyl-N'-nitrosoguanidine or nitrous acid. According to thesemethods, mutation can be introduced randomly.

With respect to a selection method for the mutant gene, recombinant DNAcomprising a DNA fragment containing the DDPS gene and vector DNA is atfirst directly subjected to a mutation treatment with hydroxylamine orthe like, which is used to transform, for example, an E. coli W3110strain. Next, transformed strains are cultivated on a minimal mediumsuch as M9 containing S-2-aminoethylcysteine (AEC) as an analog ofL-lysine. Strains harboring recombinant DNA containing the wild typeDDPS gene cannot synthesize L-lysine and diaminopimelic acid (DAP) andare suppressed in growth because DDPS expressed from the recombinant DNAis inhibited by AEC. On the contrary, a strain harboring recombinant DNAcontaining the DDPS gene in which inhibition by L-lysine is desensitizedhas a mutant enzyme encoded by the DDPS gene in the aforementionedrecombinant DNA which is not inhibited by AEC. Thus it should be capableof growth on the minimal medium in which AEC is added. This phenomenoncan be utilized to select a strain which is resistant in growth to AECas an analog of L-lysine, that is a strain harboring recombinant DNAcontaining a mutant DDPS gene in which inhibition is desensitized.

The mutant gene thus obtained may be introduced as a recombinant DNAinto a suitable host microorganism, and expressed. Thus a microorganismcan be obtained which harbors DDPS being desensitized feedbackinhibition. The host is preferably a microorganism belonging to thegenus Escherichia, for which E. coli is exemplified.

Alternatively, a mutant DDPS gene fragment may be taken out from therecombinant DNA, and inserted into another vector to make use. Thevector DNA which can be used in the present invention is preferablyplasmid vector DNA, for which there are exemplified pUC19, pUC18,pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118,pMW219 and pMW218. Besides, vectors of phage DNA can be also utilized.

Further, in order to express the mutant DDPS gene efficiently, anotherpromoter which works in microorganisms such as lac, trp and PL may beligated upstream from a DNA sequence coding for the mutant DDPS, or apromoter contained in the DDPS gene may be used as it is, or afteramplifying the promoter.

In addition, as described above, the mutant gene may be inserted into anautonomously replicable vector DNA, which is inserted into a host, andallowed to be harbored by the host as extrachromosomal DNA such as aplasmid. Alternatively, the mutant gene may be integrated intochromosome of a host microorganism by a method using transduction,transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983)),Mu phage (Japanese Patent Application Laid-open No. 2-109985) orhomologous recombination (Experiments in Molecular Genetics, Cold SpringHarbor Lab. (1972)).

<2> DNA Coding for Mutant Aspartokinase III (AKIII) Used for the PresentInvention

The DNA coding for mutant AKIII used for the present invention hasmutation to desensitize feedback inhibition of encoded AKIII by L-lysinein DNA coding for wild type AKIII. The mutation to desensitize feedbackinhibition of AKIII by L-lysine is exemplified by:

(a) mutation to replace a 323rd glycine residue with an aspartic acidresidue;

(b) mutation to replace the 323rd glycine residue with the aspartic acidresidue and replace a 408th glycine residue with an aspartic acidresidue;

(c) mutation to replace a 34th arginine residue with a cysteine residueand replace the 323rd glycine residue with the aspartic acid residue;

(d) mutation to replace a 325th leucine residue with a phenylalanineresidue;

(e) mutation to replace a 318th methionine residue with an isoleucineresidue;

(f) mutation to replace the 318th methionine residue with the isoleucineresidue and replace a 349th valine residue with a methionine residue;

(g) mutation to replace a 345th serine residue with a leucine residue;

(h) mutation to replace a 347th valine residue with a methionineresidue;

(i) mutation to replace a 352nd threonine residue with an isoleucineresidue;

(j) mutation to replace the 352nd threonine residue with the isoleucineresidue and replace a 369th serine residue with a phenylalanine residue;

(k) mutation to replace a 164th glutamic acid residue with a lysineresidue; and

(l) mutation to replace a 417th methionine residue with an isoleucineresidue and replace a 419th cysteine residue with a tyrosine residue;

as counted from the N-terminal of AKIII in an amino acid sequence ofAKIII defined in SEQ ID NO:8 in Sequence Listing.

The DNA coding for the wild type AKIII is not especially limited, forwhich DNA coding for AKIII originating from a bacterium belonging to thegenus Escherichia such as E. coli is exemplified. Concretely, there areexemplified DNA coding for an amino acid sequence defined in SEQ IDNO:8, and a sequence represented by base numbers 584-1930 in a basesequence defined in SEQ ID NO:7. Incidentally, AKIII of E. coli isencoded by a lysC gene.

In these sequences, those which have mutation in base sequence to causereplacement of amino acid residues described above are DNA coding forthe mutant AKIII of the present invention. Any codon corresponding tothe replaced amino acid residue is available especially regardless ofits kind, provided that it codes for the identical amino acid residue.Further, there are those in which amino acid sequences of possessed wildtype AKIII are slightly different depending on difference in bacterialspecies and bacterial strains. Those having replacement, deletion orinsertion of amino acid residue(s) at position(s) irrelevant to enzymeactivity in such a manner are also included in the mutant AKIII gene ofthe present invention. For example, a base sequence of a wild type lysCgene obtained in Example 2 described below (SEQ ID NO:7) is differentfrom an already published sequence of lysC of an E. coli K-12 JC411strain at 6 sites (Cassan, M., Parsot, C., Cohen, G. N., and Patte, J.C., J. Biol. Chem., 261 1052 (1986)). Encoded amino acid residues aredifferent at 2 sites of them (in lysC of the JC411 strain, a 58thglycine residue is replaced with a cysteine residue, and a 401st glycineresidue is replaced with an alanine residue, as counted from theN-terminal in an amino acid sequence of lysC defined in SEQ ID NO:8). Itis expected even for lysC having the same sequence as that of lysC ofthe E. coli K-12 JC411 strain that lysC having mutation in whichfeedback inhibition by L-lysine is desensitized is obtained if any ofthe aforementioned mutation of (a) to (1) is introduced.

A method for obtaining DNA coding for the mutant AKIII in which feedbackinhibition by L-lysine is desensitized is as follows. At first, a DNAcontaining a wild type AKIII gene or AKIII gene having another mutationis subjected to an in vitro mutation treatment, and a DNA after themutation treatment is ligated with a vector DNA adapted to a host toobtain a recombinant DNA. The recombinant DNA is introduced into a hostmicroorganism to obtain transformants. When one which expresses a mutantAKIII is selected among the aforementioned transformants, such atransformant harbors a mutant gene. Alternatively, a DNA containing awild type AKIII gene or AKIII gene having another mutation may beligated with a vector DNA adapted to a host to obtain a recombinant DNA.The recombinant DNA is thereafter subjected to an in vitro mutationtreatment, and a recombinant DNA after the mutation treatment isintroduced into a host microorganism to obtain transformants. When onewhich expresses a mutant AKIII is selected among the aforementionedtransformants, such a transformant also harbors a mutant gene.

Alternatively, it is also acceptable that a microorganism which producesa wild type enzyme is subjected to a mutation treatment to create amutant strain which produces a mutant enzyme, and then a mutant gene isobtained from the mutant strain. The agent for performing a directmutation treatment of DNA is exemplified by hydroxylamine and the like.Hydroxylamine is a chemical mutation treatment agent which causesmutation from cytosine to thymine by changing cytosine to N⁴-hydroxycytosine. Alternatively, when a microorganisms itself issubjected to a mutation treatment, the treatment is performed byultraviolet light irradiation, or using a mutating agent usually usedfor artificial mutation such as N-methyl-N'-nitro-N-nitrosoguanidine(NTG).

Any one is used as a donor microorganism for DNA containing the wildtype AKIII gene or AKIII gene having another mutation described above,provided that it is a microorganism belonging to the genus Escherichia.Concretely, it is possible to utilize those described in a book writtenby Neidhardt et al. (Neidhardt, F. C. et al., Escherichia coli andSalmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1). For example, an E. coli JM109 strain and an MC1061strain are exemplified. When the AKIII gene is obtained from thesestrains, preparation of chromosomal DNA, preparation of a chromosomalDNA library and the like may be performed in the same manner as thepreparation of the DDPS gene described above. As the host to be used forpreparation of the library, it is preferable to use a strain entirelydeficient in AKI, II and III such as an E. coli GT3 strain (availablefrom E. coli Genetic Stock Center (Connecticut, United States)).

From the obtained chromosomal DNA library, a bacterial strain having arecombinant DNA of the AKIII gene is obtained as a strain in which theAKIII activity is increased, or a strain in which auxotrophy iscomplemented. Cellular extracts are prepared from candidate strains, andcrude enzyme solutions are prepared therefrom to confirm the AKIIIactivity. The measurement procedure for the AKIII enzyme activity may beperformed in accordance with a method of Stadtman et al. (Stadtman, E.R., Cohen, G. N., LeBras, G., and Robichon-Szulmajster, H., J. Biol.Chem., 236, 2033 (1961)).

For example, when a mutant strain completely deficient in AK is used asa host, a DNA fragment containing an AKIII gene can be obtained byisolating a transformed strain which becomes capable of growing on amedium not containing L-lysine, L-threonine, L-methionine anddiaminopimelic acid, or on a medium not containing homoserine anddiaminopimelic acid, and recovering recombinant DNA from the bacterialstrain.

When the AKIII gene is amplified from chromosomal DNA by means of thePCR method, DNA primers to be used for the PCR reaction can be properlyprepared on the basis of, for example, a sequence known in E. coli(Cassan, M., Parsot, C., Cohen, G. N., and Patte, J. C., J. Biol. Chem.,261, 1052 (1986)). However, primers which can amplify a regioncomprising 1347 bases coding for lysC gene is suitable, and for example,two primers having sequences defined in SEQ ID NO:5 and NO:6 aresuitable.

The method for carrying out mutation such as replacement, insertion anddeletion of amino acid residue(s) on the AKIII gene obtained asdescribed above is exemplified by the recombinant PCR method, the sitespecific mutagenesis method and the like, in the same manner as themutation treatment of the DDPS gene described above.

Further, according to chemical synthesis of an aimed gene, it ispossible to introduce mutation or random mutation into an aimed site.

Further, a method is available in which DNA of the AKIII gene onchromosome or extrachromosomal recombinant DNA is directly treated withhydroxylamine (Hashimoto, T. and Sekiguchi, M. J. Bacteriol., 159, 1039(1984)). Alternatively, it is acceptable to use a method in which abacterium belonging to the genus Escherichia having an AKIII gene onchromosome or extrachromosomal recombinant DNA is irradiated byultraviolet light, or a method to perform a treatment with a chemicalagent such as N-methyl-N'-nitrosoguanidine or nitrous acid.

With respect to a selection method for the mutant AKIII gene, a straincompletely deficient in AK, for example, an E. coli GT3 strain is atfirst transformed with a recombinant DNA containing an AKIII gene havingbeen subjected to the mutation treatment. Next, transformed strains arecultivated on a minimal medium such as M9 containing a considerableamount of L-lysine. Strains harboring recombinant DNA containing a wildtype AKIII gene cannot synthesize L-threonine, L-isoleucine,L-methionine and diaminopimelic acid (DAP) and are suppressed in growthbecause only one AK is inhibited by L-lysine. On the contrary, thestrain harboring recombinant DNA containing the mutant AKIII gene inwhich inhibition by L-lysine is desensitized should be capable of growthon the minimal medium added with the considerable amount of L-lysine.This phenomenon can be utilized to select a strain which is resistant ingrowth to L-lysine or AEC as an analog of L-lysine, that is a strainharboring recombinant DNA containing a mutant AKIII gene in whichinhibition is desensitized.

The mutant gene thus obtained may be introduced as a recombinant DNAinto a suitable microorganism (host), and expressed. Thus amicroorganism can be obtained which harbors AKIII being desensitizedfeedback inhibition.

The host is preferably a microorganism belonging to the genusEscherichia, for which E. coli is exemplified.

Alternatively, a mutant AKIII gene fragment may be taken out from therecombinant DNA, and inserted into another vector to make use. Thevector DNA which can be used in the present invention is preferablyplasmid vector DNA, for which there are exemplified pUC19, pUC18,pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118,pMW219 and pMW218. Besides, vectors of phage DNA can be also utilized.

Further, in order to express the mutant AKIII gene efficiently, anotherpromoter which works in microorganisms such as lac, trp and PL may beligated upstream from a DNA sequence coding for the mutant AKIII, or apromoter contained in the AKIII gene may be used as it is, or afteramplifying it.

In addition, as described above, the mutant gene may be inserted into anautonomously replicable vector DNA, inserted into a host, and allowed tobe harbored by the host as extrachromosomal DNA such as plasmid.Alternatively, the mutant gene may be integrated into chromosome of ahost microorganism by a method using transduction, transposon (Berg, D.E. and Berg, C. M., Bio/Technol., 1, 417 (1983)), Mu phage (JapanesePatent Application Laid-open No. 2-109985) or homologous recombination(Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).

<3> Production of L-lysine According to the Present Invention

L-lysine can be efficiently produced by cultivating, in a preferredmedium, the bacterium transformed by introducing the mutant DDPS geneobtained as described above and allowed to harbor AK which isdesensitized feedback inhibition by L-lysine, producing and accumulatingL-lysine in a culture thereof, and collecting L-lysine from the culture.Namely, L-lysine can be efficiently produced by allowing the bacteriumbelonging to the genus Escherichia to harbor both of the mutant DDPS andthe mutant AKIII.

The bacterium belonging to the genus Escherichia harboring AK which isdesensitized feedback inhibition by L-lysine is exemplified by bacteriabelonging to the genus Escherichia transformed by integrating, intochromosomal DNA, a DNA coding for AKIII having mutation to desensitizefeedback inhibition by L-lysine, or bacteria belonging to the genusEscherichia transformed by introducing, into cells, a recombinant DNAconstructed by ligating the DNA with a vector DNA autonomouslyreplicable in cells of bacteria belonging to the genus Escherichia.Further, AK in which feedback inhibition by L-lysine is desensitized maybe a wild type AK which does not suffer feedback inhibition by L-lysine,or one to which such a wild type AK gene is introduced into a bacteriumbelonging to the genus Escherichia in the same manner. Further, a mutantstrain of a bacterium belonging to the genus Escherichia, which hasbecome to produce a mutant AKIII by means of a mutation treatment ofcells of a bacterium belonging to the genus Escherichia, is alsoacceptable.

On the other hand, in order to achieve transformation by introducing themutant DDPS gene into a bacterium belonging to the genus Escherichia,the mutant DDPS gene may be integrated into chromosomal DNA to achievetransformation, or transformation may be achieved by introducing, intocells, a recombinant DNA constructed by ligating the mutant DDPS genewith a vector DNA autonomously replicable in cells of bacteria belongingto the genus Escherichia.

When the both of the mutant DDPS gene and the mutant AKIII gene areintroduced into a bacterium belonging to the genus Escherichia, the bothmutant genes may be integrated into and harbored on chromosomal DNA ofthe bacterium belonging to the genus Escherichia, or they may beharbored on an identical plasmid or separated plasmids in cells asextrachromosomal DNA. When separated plasmids are used, it is preferableto use plasmids having a stable distribution mechanism to allow each ofthem to be compatibly harbored in the cell. Further, one of the mutantgenes may be integrated into and harbored on chromosomal DNA, and theother mutant gene may be harbored on a plasmid in cells asextrachromosomal DNA, respectively. When the mutant DDPS gene and themutant AKIII gene are introduced into a bacterium belonging to the genusEscherichia, any order of introduction of the both genes is acceptable.

The productivity of L-lysine can be further improved by enhancing adihydrodipicolinate reductase gene of the bacterium belonging to thegenus Escherichia in which the mutant DDPS gene and the mutant AKIIIgene have been introduced. The productivity of L-lysine can be stillfurther improved by introducing a diaminopimelate dehydrogenase geneoriginating from a coryneform bacterium into the bacterium belonging tothe genus Escherichia in which the dihydrodipicolinate reductase genehas been enhanced. This diaminopimelate dehydrogenase gene should beenhanced. Alternatively, the productivity of L-lysine can be alsoimproved in a similar degree by enhancing tetrahydrodipicolinatesuccinylase gene and a succinyldiaminopimelate deacylase gene instead ofthe introduction of the diaminopimelate dehydrogenase.

The enhancement of gene herein refers to enhancement in activity of anenzyme as an expression product of the gene per a cell. Concretely,there may be exemplified enhancement in copy number of the gene in acell, enhancement in expression amount per the gene by using a promoterhaving a high expression efficiency, and introduction of mutation toenhance enzyme activity into the gene. In order to enhance the copynumber of a gene in a cell, the gene is inserted into a vectorautonomously replicable in bacteria belonging to the genus Escherichia,and a bacterium belonging to the genus Escherichia may be transformedwith this vector. This vector is preferably a multi-copy type plasmid.Alternatively, the copy number may be increased by amplifying DNAintegrated into chromosomal DNA by using Mu phage or the like. Withrespect to the use of the plasmid, when plasmids are used forintroduction of the mutant DDPS gene and the mutant AKIII gene, suchplasmids having a stable distribution mechanism are preferably used inwhich these plasmids are stably harbored in a cell together. Any orderof introduction of the genes is acceptable.

A mechanism will be explained below in which the productivity ofL-lysine can be improved in a stepwise manner by successively enhancinggenes of the L-lysine biosynthesis system as described above. Abiosynthesis system comprising a plurality of reactions can be comparedto a liquid flowing through a plurality of conduits having differentthicknesses connected in serial. Herein each conduit corresponds to anindividual enzyme, and the thickness of the conduit corresponds to anenzyme reaction velocity. In order to increase the amount of the liquidflowing through the conduits, it is effective to thicken the thinnestpipe. No effect can be expected even if a thick conduit is furtherthickened. In order to further increase the flow amount, the secondthinnest conduit may be thickened. From such a viewpoint, the presentinventors have tried to enhance the L-lysine biosynthesis system. Forthis purpose, as shown in Example 6 described below, the order of ratedetermining steps of the L-lysine biosynthesis system has beenelucidated by introducing, into E. coli, genes of the L-lysinebiosynthesis system originating from E. coli in a stepwise manner. Inthis elucidation, four genes of dapC succinyldiaminopimelatetransaminase dapD (tetrahydrodipicolinate succinylase gene) dapE(succinyldiaminopimelate deacylase gene), and dapF (diaminopimelateepimerase gene) located downstream in the biosynthesis pathway werereplaced with a gene DDH coding for DDH (diaminopimelate dehydrogenase)of Brevibacterium lactofermentum capable of catalyzing reactionsparticipated by these gene products by itself. Namely, introduced genesfor enzymes of the L-lysine biosynthesis system and the enzymes encodedby them are as follows:

ppc: phosphoenolpyruvate carboxylase

aspC: aspartate aminotransferase

lysC: aspartokinase III

lysC*: inhibition-desensitized aspartokinase III

asd: aspartate semialdehyde dehydrogenase

dapA: dihydrodipicolinate synthase

dapA*: inhibition-desensitized dihydrodipicolinate synthase

dapB: dihydrodipicolinate reductase

DDH: diaminopimelate dehydrogenase (originating from Brevibacteriumlactofermentum)

lysA: diaminopimelate decarboxylase

As a result of individual introduction of each of the genes into E.coli, production of L-lysine was found in strains in which lysC*, dapAor dapA* was introduced, and a dapA*-introduced strain showed thehighest L-lysine productivity. According to the result, it was foundthat a reaction catalyzed by dapA was the first rate determining step.Next, when each of the genes of the L-lysine biosynthesis system wasintroduced into the dapA*-introduced strain, lysC* had the largesteffect on the improvement in L-lysine productivity. Thus it was foundthat a reaction catalyzed by lysC was the second rate determining step.In the same manner, it was found that a reaction catalyzed by dapB wasthe third rate determining step, and a reaction catalyzed by DDH was thefourth rate determining step. Further, as a result of investigation onrate determining steps among reactions catalyzed by dapC, dapD, dapE anddapF replaced with DDH, it was found that dapD and dapE concerned ratedetermining.

A method for obtaining the genes of the L-lysine biosynthesis system ofE. coli and the DDH gene of Brevibacterium lactofermentum will beexemplified below.

The ppc gene can be obtained from a plasmid pS2 (Sabe, H. et al., Gene,31, 279 (1984)) or pT2 having this gene. A DNA fragment containing theppc gene is obtained by cutting pS2 with AatII and AflII. A DNA fragmenthaving the ppc gene is also obtained by cutting pT2 with SmaI and ScaI.An E. coli F15 strain (AJ12873) harboring pT2 is internationallydeposited in National Institute of Bioscience and Human Technology ofAgency of Industrial Science and Technology (postal code: 305, 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) under a depositionnumber of FERM BP-4732 based on the Budapest Treaty.

The aspc gene is obtained from a plasmid pLF4 (Inokuchi, K. et al.,Nucleic Acids Res., 10, 6957 (1982)) having this gene. A DNA fragmenthaving the aspc gene is obtained by cutting pLF4 with PvuII and StuI.

The asd gene is obtained from a plasmid pAD20 (Haziza, C. et al., EMBO,1, 379 (1982)) having this gene. A DNA fragment having the asd gene isobtained by cutting pAD20 with AseI and ClaI.

The dapB gene is obtained by amplifying chromosomal DNA of E. coli bymeans of the PCR method by using two species of oligonucleotide primers(for example, SEQ ID NO:9, NO:10) prepared on the basis of a nucleotidesequence of a known dapB gene (Bouvier, J. et al., J. Biol. Chem., 259,14829 (1984)).

The DDH gene is obtained by amplifying chromosomal DNA of Brevibacteriumlactofermentum by means of the PCR method by using two species ofoligonucleotide primers (for example, SEQ ID NO:11, NO:12) prepared onthe basis of a known nucleotide sequence of a DDH gene ofCorynebacterium glutamicum (Ishino, S. et al., Nucleic Acids Res., 15,3917 (1987)).

The lysA gene is obtained by amplifying chromosomal DNA of E. coli bymeans of the PCR method by using two species of oligonucleotide primers(for example, SEQ ID NO:13, NO:14) prepared on the basis of a nucleotidesequence of a known lysA gene (Stragier, P. et al., J. Mol. Biol., 168,321 (1983)).

The dapD gene is obtained by amplifying chromosomal DNA of an E. coliW3110 strain by means of the PCR method by using two species ofoligonucleotide primers (for example, SEQ ID NO:15, NO:16) prepared onthe basis of a nucleotide sequence of a known dapD gene (Richaud, C. etal., J. Biol. Chem., 259, 14824 (1984)).

The dapE gene is obtained by amplifying E. coli DNA by means of the PCRmethod by using two species of oligonucleotide primers (SEQ ID NO:17,NO:18) prepared on the basis of a nucleotide sequence of a known dapEgene (Bouvier, J. et al., J. Bacteriol., 174, 5265 (1992)).

The dapF gene is obtained by amplifying chromosomal DNA of E. coli bymeans of the PCR method by using two species of oligonucleotide primers(for example, SEQ ID NO:19, NO:20) prepared on the basis of a nucleotidesequence of a known dapF gene (Richaud, C. et al., Nucleic Acids Res.,16, 10367 (1988)).

In the present invention, any bacterium belonging to the genusEscherichia is available for the use as a host provided that a promoterof the mutant DDPS gene, the mutant AKIII gene or another gene of theL-lysine biosynthesis system, or another promoter for expressing thesegenes functions in its cells, and a replication origin of a vector DNAto be used for introduction functions in its cells to be capable ofreplication when the mutant DDPS gene, the mutant AKIII gene or anothergene of the L-lysine biosynthesis system is introduced into a plasmid asextrachromosomal DNA.

For example, there may be exemplified L-lysine-producing E. coli,concretely a mutant strain having resistance to L-lysine analogs. Thelysine analog is such one which inhibits proliferation of bacteriabelonging to the genus Escherichia, but the suppression is entirely orpartially desensitized if L-lysine co-exists in a medium. For example,there are oxalysine, lysine hydroxamate, AEC, γ-methyllysine,α-chlorocaprolactam and the like. Mutant strains having resistance tothese lysine analogs are obtained by applying an ordinary artificialmutation operation to microorganisms belonging to the genus Escherichia.The bacterial strain to be used for L-lysine production is concretelyexemplified by Escherichia coli AJ11442 (deposited as FERM BP-1543 andNRRL B-12185; see Japanese Patent Application Laid-open No. 56-18596 orU.S. Pat. No. 4,346,170). In aspartokinase of the microorganismsdescribed above, feedback inhibition by L-lysine is desensitized.

Besides, for example, L-threonine-producing microorganisms areexemplified, because inhibition of their aspartokinase by L-lysine isgenerally desensitized also in the L-threonine-producing microorganisms.As an L-threonine-producing bacterium belonging to E. coli, a B-3996strain has the highest producibility known at present. The B-3996 strainis deposited in Research Institute for Genetics and IndustrialMicroorganism Breeding under a registration number of RIA 1867.

The medium to be used for cultivation of the transformant harboring themutant gene according to the present invention is an ordinary mediumcontaining a carbon source, a nitrogen source, organic ions andoptionally other organic components.

As the carbon source, it is possible to use sugars such as glucose,lactose, galactose, fructose, or starch hydrolysate; alcohols such asglycerol or sorbitol; or organic acids such as fumaric acid, citric acidor succinic acid.

As the nitrogen source, it is possible to use inorganic ammonium saltssuch as ammonium sulfate, ammonium chloride or ammonium phosphate;organic nitrogen such as soybean hydrolysate; ammonia gas; or aqueousammonia.

It is desirable to allow required substances such as vitamin B₁ andL-isoleucine or yeast extract to be contained in appropriate amounts asorganic trace nutrients. Other than the above, potassium phosphate,magnesium sulfate, iron ion, manganese ion and the like are added insmall amounts, if necessary.

Cultivation is preferably carried out under an aerobic condition for16-72 hours. The cultivation temperature is controlled at 25° C. to 45°C., and pH is controlled at 5-8 during cultivation. Inorganic ororganic, acidic or alkaline substances as well as ammonia gas or thelike can be used for pH adjustment.

Collection of L-lysine from a fermented liquor is usually carried out bycombining an ion exchange resin method, a precipitation method and otherknown methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows preparation steps for pdapA1 and pdapA2.

FIG. 2 shows inhibition by L-lysine for wild type and mutant DDPS's.

FIG. 3 shows preparation steps for a plasmid pdapAS824 having a doublemutation type dapA* gene.

FIG. 4 shows preparation steps for pLYSC1 and pLYSC2.

FIG. 5 shows an appearance ratio and a mutation ratio of transformantsafter a hydroxylamine treatment.

FIG. 6 shows inhibition by L-lysine for wild type and mutant AKIII's.

FIG. 7 shows preparation steps for a plasmid RSF24P originating fromRSF1010 having dapA*24.

FIG. 8 shows preparation steps for a plasmid pLLC*80.

FIG. 9 shows preparation steps for a plasmid RSFD80 originating fromRSF1010 having dapA*24 and lysC*80.

FIG. 10 shows structures of plasmids pdapA and pdapA* having dapA ordapA*.

FIG. 11 shows structures of plasmids plysC and plysC* having lysC orlysC*80.

FIG. 12 shows a structure of a plasmid pppc having ppc.

FIG. 13 shows a structure of a plasmid paspc having aspc.

FIG. 14 shows a structure of a plasmid pasd having asd.

FIG. 15 shows a structure of a plasmid pdapB having dapB.

FIG. 16 shows a structure of a plasmid pDDH having DDH.

FIG. 17 shows a structure of a plasmid plysA having lysA.

FIG. 18 shows preparation steps for a plasmid pCAB1 originating fromRSF1010 having dapA*24, lysC*80 and dapB.

FIG. 19 shows preparation steps for a plasmid pCABD2 originating fromRSF1010 having dapA*24, lysC*80, dapB and DDH.

FIG. 20 shows a structure of a plasmid pdapD having dapD.

FIG. 21 shows a structure of a plasmid pdapE having dapE.

FIG. 22 shows a structure of a plasmid pdapF having dapF.

FIG. 23 shows preparation steps for a plasmid pMWdapDE1 having dapD anddapE.

FIG. 24 shows preparation steps for a plasmid pCABDE1 having dapA*24,lysC*80, dapB, dapD and dapE.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more concretely explained below withreference to Examples.

EXAMPLE 1

Preparation of Mutant DDPS Gene

<1> Cloning of Wild Type dapA Gene

A nucleotide sequence of a dapA gene of E. coli has been alreadyreported (Richaud, F. et al., J. Bacteriol., 297 (1986)), and it isknown that its open reading frame (ORF) comprises 876 base pairs, andcodes for a protein comprising 292 amino acid residues. Since it isunknown how this dapA gene is regulated, a region containing only an SDsequence and ORF except for a promoter region was amplified by using thePCR method and cloned.

Total genomic DNA of an E. coli K-12 MC1061 strain was extracted inaccordance with a method of Saito and Miura (Biochem. Biophys. Acta.,72, 619 (1963)). Two species of primers having sequences shown in SEQ IDNO:1 and NO:2 were prepared, which were used to perform the PCR reactionin accordance with a method of Erlich et al. (PCR Technology, Stocktonpress (1989)), and target DNA was amplified. Obtained DNA was insertedinto a commercially available cloning vector pCR1000 for PCR fragments(purchased from Invitrogen, Ltd., (California, the United States)) as itwas. pCR1000 contains a lacZ promoter (Placz), and is sold in a state ofbeing cut at a site downstream from the lacZ promoter. When arecombinant DNA obtained by ligating a PCR fragment between both cuttermini of pCR1000 is introduced into E. coli, the PCR fragment istranscribed under control of the lacZ promoter. Upon ligation of the PCRfragment with PCR1000, two species of plasmids were obtained, which werepdapA1 as a plasmid ligated in a normal orientation and pdapA2 as aplasmid ligated in a reversed orientation, for the direction oftranscription of dapA with respect to the direction of transcription bythe lacZ promoter (FIG. 1).

When these plasmids were introduced into E. coli JE7627 which is astrain deficient in DDPS, strains with the introduced plasmids iscomplemented auxotrophy for diaminopimelic acid of the host JE7627. Thusit was confirmed that DNA fragments inserted into the both plasmidscontain the gene dapA coding for active DDPS.

A transformed strain obtained by introducing pdapA1 into a wild type E.coli W3110 strain (available from National Institute of Genetics(Mishima-shi, Shizuoka-ken, Japan)) was designated as W3110/pdapA1, anda transformed strain obtained by introducing pdapA2 into the E. coliW3110 strain was designated as W3110/pdapA2, respectively. These twotransformed strains were cultivated respectively in a minimal medium M9having the following composition added with AEC as an analog of lysine.The W3110 strain with no introduced plasmid was also cultivated in thesame medium as a control. These two transformed strains and the W3110strain having no plasmid were suppressed in growth by AEC, however,their growth inhibition was recovered by addition of L-lysine.

(Minimal medium M9)

    ______________________________________                                        A:          (20 × M9)                                                                             Na                                                                          .sub.2 HPO.sub.4 ·12H.sub.2 O 303 g/L                                  KH.sub.2 PO.sub.4 60 g/L                               NaCl 10 g/L                                                                   NH.sub.4 Cl 20 g/L                                                         ______________________________________                                    

B: 1 M MgSO₄

C: 50% Glucose

D: 1 g/L Thiamine

A, B, C and D described above were separately sterilized, and mixed in aratio of A:B:C:D: water=5:0.1:1:0.1:95.

<2> Preparation of Mutant DDPS Gene (dapA*)

It was assumed that a strain harboring a plasmid containing dapA* codingfor DDDPS with desensitized inhibition by L-lysine could grow on aminimal medium M9 added with a considerable amount of AEC. A strainharboring a plasmid containing dapA* was selected by their growthresistance to AEC.

In order to efficiently obtain dapA*, dapA's on pdapA1 and pdapA2prepared in <1> were subjected to a mutation treatment.

(1-2-1) Investigation on Selection Condition for Strain HarboringPlasmid Containing dapA*

The W3110/pdapA1 strain and the W3110/pdapA2 strain obtained asdescribed above were cultivated on M9 agar plate media containingvarious concentrations of AEC, respectively. Growth inhibitoryconcentrations by AEC were examined, and a selection condition wasinvestigated for a strain harboring a plasmid containing dapA*.

Growth of the transformants on the M9 media containing AEC at variousconcentrations is shown in Table 1. In this table, +indicates growth oftransformant, and-indicates no growth.

                  TABLE 1                                                         ______________________________________                                        AEC concentration                                                                            W3110/pdapA1                                                                             W3110/pdapA2                                        ______________________________________                                        (mM)                                                                            250 -  -                                                                      125 - -                                                                       60 - -                                                                        30 - -                                                                        15 + -                                                                        8 + +                                                                         4 + +                                                                         2 + +                                                                       ______________________________________                                    

The direction of transcription of the dapA gene on pdapA1 coincides withthe direction of transcription by the lacZ promoter (FIG. 1). Thus itwas found that the dapA gene on pdapA1 provided resistance to AEC atconsiderably high concentrations even when dapA remained as a wild typebecause its expression amount was amplified by the lacZ promoter, whilethe dapA gene on pdapA2 had a smaller expression amount and providedinhibition in growth by AEC at lower concentrations because thedirection of transcription was in the reversed direction with respect tothe lacZ promoter, and a promoter of dapA's own was also deficient (thegrowth was suppressed in an allotment of addition of 30 mM in the caseof the W3110/pdapA1 strain, and of 15 mM in the case of the W3110/pdapA2strain). It was confirmed that the growth inhibition was eliminated bysimultaneous addition of L-lysine.

Therefore, pdapA2 was used as an object for introduction of mutation. Amedium prepared by adding 60 mM of AEC to the minimal medium M9 was usedfor selection of a strain harboring a plasmid containing dapA*. Thismedium is referred to as "selection medium" in Example 1 below.

(1-2-2) In Vitro Mutation Treatment for pdapA2 with Hydroxylamine

An in vitro mutation treatment method in which plasmids are directlytreated with hydroxylamine was used for introduction of mutation intothe pdapA2 plasmid.

2 μg of DNA was treated at 75° C. for 1-4 hours in 0.4 M hydroxylamine(0.1 M KH₂ PO₄ -1 mM EDTA (pH 6.0): 100 μl, 1 M hydroxylamine-1 mM EDTA(pH 6.0): 80 μl, DNA: 2 μg, total: 200 μl by filling up with water). DNAafter the treatment was purified with glass powder, introduced into E.coli W3110, and spread on a complete medium (L-broth: 1% Bacto trypton,0.5% Yeast extract, 0.5% NaCl, 1.5% agar), and colonies were formed.They were replicated onto the selection medium described in (1-2-1), andthose which formed colonies on the selection medium were selected.Candidates of mutant plasmids in a total of 36 strains were obtainedafter two times of experiments.

The candidate strains of 36 strains in total thus obtained were spottedon the selection medium again, and AEC resistance was confirmed.

(1-2-3) Isolation of dapA* Gene and Investigation on dapA* Product

Mutant pdapA2's were recovered from the 36 strains described above. AdapA-deficient strain, JE7627 was transformed with them and the wildtype pdapA2, respectively. A cell-free extract was prepared from each ofthe transformed strains, and the enzyme activity of DDPS was measured.

The cell-free extract (crude enzyme solution) was prepared as follows. Atransformed strain was cultivated in a 2×TY medium (1.6% Bacto trypton,1% Yeast extract, 0.5% NaCl), and collected at an optical density at 660nm (OD₆₆₀) of about 0.8. A cell pellet was washed with 0.85% NaCl undera condition of 0° C., and suspended in 20 mM potassium phosphate buffer(pH 7.5) containing 400 mM KCl. The cells were ruptured by sonication(0° C., 200 W, 10 minutes). A ruptured cell solution was centrifuged at33 krpm for 1 hour under a condition of 0° C. to obtain a supernatant towhich ammonium sulfate was added to give 80% saturation to be stored at0° C. overnight followed by centrifugation. A pellet was dissolved in 20mM potassium phosphate buffer (pH 7.5)-400 mM KCl.

The enzyme activity of DDPS was measured in accordance with a method ofYugari et al. (Yugari, Y. and Gilvarg, C., J. Biol. Chem., 240, 4710(1962)). Namely, the absorbance of a reaction solution having thefollowing composition was measured at 37° C. with a spectrophotometer ata wavelength of 270 nm in a time-dependent manner. And generateddihydrodipicolinate was measured. Sodium pyruvate was removed from thereaction system to be used as a blank.

(Composition of Reaction Solution)

50 mM imidazole-HCl pH 7.4

20 mM L-aspartate semialdehyde

20 mM sodium pyruvate

enzyme solution

water (balance)

total 1.0 ml

Various concentrations of L-lysine were added to the enzyme reactionsolution during measurement of the enzyme activity of DDPS, and thedegree of inhibition by L-lysine was examined. As shown in FIG. 2, thewild type DDPS suffered inhibition by L-lysine. Mutant plasmidsoriginating from the transformed strains having DDPS difficult to sufferinhibition by L-lysine as compared with the wild type were three speciesamong the 36 species of the candidate plasmids. They were designated aspdapAS8, pdapAS9 and pdapAS24, respectively. According to followingdetermination of nucleotide sequences, it was revealed that pdapAS8 andpdapAs9 had the same mutation.

The degree of desensitization of inhibition by L-lysine was varied inthe three species of mutant DDPS encoded by pdapAS8, pdapAS9 andpdapAS24, however, the inhibition by L-lysine was desensitized in all ofthe three species. Although the specific activity of the enzyme might beaffected by growth situations of cells and preparation of samples, itwas found to be lowered a little in any case as compared with the wildtype. However, it was judged that no substantial problem would be causedby them as a material for breeding.

(1-2-4) Determination of Nucleotide Sequence of Mutant dapA Gene

Nucleotide sequences of the mutant dapA genes were determined inaccordance with an ordinary method by using a DNA sequencer ABI Model373A (produced by Applied Biosystems Inc.). As a result, it was revealedthat 487th C was changed to T in pdapAS8 and pdapAS9, and 597th C waschanged to T in pdapAS24 on a sequence of the wild type dapA gene shownin SEQ ID NO:3. Therefore, it was revealed that a 81st alanine residuewas changed to a valine residue in DDPS encoded by pdapAS8 and pdapAS9,and a 118th histidine residue was changed to a tyrosine residue in DDPSencoded by pdapAS24 in an amino acid sequence of DDPS shown in SEQ IDNO:4.

(1-2-5) Preparation of dapA Having Double Mutation

Two species of the mutant dapA genes were obtained as described above.In order to verify whether or not desensitization of inhibition worksadditively for these mutations, a plasmid containing mutant dapA havingboth of the two mutations was prepared. A procedure of preparation is asshown in FIG. 3. An obtained plasmid having double mutation wasdesignated as pdapAS824.

EXAMPLE 2

Preparation of Mutant AKIII Gene

<1> Cloning of Wild Type lysC Gene

A nucleotide sequence of an AKIII gene (lysC) of E. coli has beenalready reported (Cassan, M., Parsot, C., Cohen, G. N., and Patte, J.C., J. Biol. Chem., 261, 1052 (1986)), and it is known that its openreading frame (ORF) comprises 1347 base pairs, and codes for a proteincomprising 449 amino acid residues. An operator is present in this gene,and is subjected to suppression by L-lysine. Thus in order to remove theoperator region, a region containing only an SD sequence and ORF wasamplified by using the PCR method and cloned.

Total genomic DNA of an E. coli K-12 MC1061 strain was prepared inaccordance with a method of Saito and Miura (Biochem. Biophys. Acta.,72, 619 (1963)). Two species of primers having sequences shown in SEQ IDNO:5 and NO:6 were prepared, which were used to perform the PCR reactionin accordance with a method of Erlich et al. (PCR Technology, Stocktonpress (1989)), and the lysc gene was amplified. Obtained DNA wasdigested with BamHI and AseI, then blunt-ended, and inserted into a SmaIsite of a multi-copy vector, pUC18. This SmaI site is located at adownstream side from a lacZ promoter existing in the vector, and whenrecombinant DNA obtained by inserting a DNA fragment into the SmaI siteof pUC18 is introduced into E. coli, the inserted DNA fragment istranscribed by means of read-through transcription under the control bythe lacZ promoter. Upon insertion of the PCR fragment into the SmaI siteof pUC18, two species of plasmids were obtained, which were pLYSC1 as aplasmid inserted in a reversed orientation and pLYSC2 as a plasmidinserted in a normal orientation, for the direction of transcription oflysC with respect to the direction of transcription by the lacZ promoter(FIG. 4).

When these plasmids were used to transform E. coli GT3 (thrA1016b,metLM1005, lysC1004) as a completely deficient strain for AKI, II, III,auxotrophy of GT3 for homoserine and diaminopimelic acid wascomplemented. Thus it was confirmed that DNA fragments inserted into theboth plasmids contain the gene lysC coding for active AKIII.

A transformed strain obtained by introducing pLYSC1 into the AKcompletely deficient strain, E. coli GT3 was designated as GT3/pLYSC1,and a transformed strain obtained by introducing pLYSC2 into the E coliGT3 was designated as GT3/pLYSC2. A considerable amount of L-lysine wasadded to the minimal medium M9, and the GT3/pLYSC1 strain and theGT3/pLYSC2 strain were cultivated, respectively. Both of the GT3/pLYSC1strain and the GT3/pLYSC2 strain harbor plasmids containing the wildtype lysC, in which AKIII encoded by lysC on the plasmids is a sole AK.The wild type AKIII as the sole AK is inhibited by L-lysine in thepresence of a considerable amount of L-lysine. Thus the both strainscould not synthesize L-threonine, L-isoleucine, L-methionine anddiaminopimelic acid (DAP), and were suppressed in growth.

<2> Preparation of Mutant AKIII Gene (lysC*)

It was assumed that a strain harboring a plasmid containing lysc* codingfor AK with desensitized inhibition by L-lysine could grow on a minimalmedium M9 added with a considerable amount of L-lysine. A strainharboring a plasmid containing lysC* was selected by selecting strainswith their growth resistant to L-lysine or AEC as an analog of L-lysine.

In order to efficiently obtain lysc*, lysc's on pLYSC1 and pLYSC2prepared in <1> were subjected to a mutation treatment.

(2-2-1) Investigation on Selection Condition for Strain HarboringPlasmid Containing lysC*

The GT3/pLYSC1 strain and the GT3/pLYSC2 strain were cultivated on M9agar plate media containing various concentrations of L-lysine or AEC,respectively. Growth inhibitory concentrations by L-lysine or AEC wereexamined, and a selection condition was investigated for a strainharboring a plasmid containing lysC*.

Growth of the transformants on the M9 media containing L-lysine or AECat various concentrations is shown in Table 2. In this table, +indicates growth of transformant, ± indicates a little growth, and -indicates no growth.

                                      TABLE 2                                     __________________________________________________________________________    Growth and L-lysine concentration                                                    0  0.2                                                                             0.4                                                                             0.8                                                                             1.5                                                                             3 6 12                                                                              25                                                                              50                                                                              100                                                                              200                                                                              (mM)                                        __________________________________________________________________________      GT3/pLYSC1 +  - - - - - - - - - - -                                           GT3/pLYSC2 + + + + + + + + + + + -                                          Growth and AEC concentration                                                         0 0.2                                                                              0.4                                                                              0.8                                                                              1.5                                                                              3  6  12 25                                                                              50                                                                              (mM)                                        __________________________________________________________________________      GT3/pLYSC1 +  - - - - - - - - -                                               GT3/pLYSC2 + ± ± ± ± ± - - - -                               __________________________________________________________________________

The direction of transcription of the lysC gene on pLYSC2 coincides withthe direction of transcription by the lacZ promoter (FIG. 4). Thus itwas found that the lysc gene on pLYSC2 provided resistance to L-lysineand AEC at considerably high concentrations even when lysC remained as awild type because its expression amount was amplified by the lacZpromoter, while the lysC gene on pLYSC1 had a smaller expression amountand provided inhibition in growth by L-lysine and AEC at lowerconcentrations because the direction of transcription was in thereversed direction with respect to the lacZ promoter, and a promoter ofitself was also deficient (the growth was not suppressed up to anallotment of addition of 100 mM for L-lysine and up to an allotment ofaddition of 3 mM for AEC in the case of the GT3/pLYSC2 strain, while thegrowth was completely suppressed in an allotment of addition of 0.2 mMfor both L-lysine and AEC in the case of GT3/pLYSC1 strain). It wasconfirmed that the growth inhibition was eliminated by simultaneousaddition of homoserine and diaminopimelic acid.

Therefore, pLYSC1 was used for experiments of introduction of mutation.A medium prepared by adding 10 mM of L-lysine or 0.2 mM of AEC to theminimal medium M9 was used for selection of plasmid-harboring strainscontaining lysC*. This medium is referred to as "selection medium" inExample 2 below.

(2-2-2) In Vitro Mutation Treatment for pLYSC1 with Hydroxylamine

Two kinds of methods were used for introduction of mutation into thepLYSC1 plasmid, which were an in vitro mutation treatment method inwhich plasmids are directly treated with hydroxylamine, and anadditional in vivo mutation treatment method in which a cell harboring aplasmid is treated with nitrosoguanidine (NTG) followed by extraction ofthe plasmid in order to provide diversity of mutation, namely expectingmutation other than the mutation from cytosine to thymine withhydroxylamine.

(In Vitro Mutation Treatment with Hydroxylamine)

2 μg of DNA was treated under a condition of 75° C. for 1-4 hours in 0.4M hydroxylamine (0.1 M KH₂ PO₄ -1 mM EDTA (pH 6.0): 100 μl, 1 Mhydroxylamine-1 mM EDTA (pH 6.0): 80 μl, DNA: 2 μg, total: 200 μl byfilling up with water). DNA after the treatment was purified with glasspowder, introduced into an AK completely deficient strain, an E. coliGT3 strain, and spread on a complete medium (L-broth: 1% Bacto trypton,0.5% Yeast extract, 0.5% NaCl, 1.5% agar), and colonies were formed.They were replicated onto the selection medium described in (2-2-1), andstrains capable of growth on the selection medium were selected ascandidate strains. The appearance ratio of transformants and themutation ratio were found to proceed as shown in FIG. 5. Mutant strainswere obtained by a treatment for 4 hours at a considerably high ratio of0.5-0.8%.

(In Vivo Mutation Treatment with NTG)

pLYSC1 was introduced into E. coli MC1061, and an NTG treatment wasperformed with a whole cell. The cell after the treatment was cultivatedovernight to fix mutation, and then a plasmid was extracted andintroduced into E. coli GT3. Namely, the transformed strain wascultivated in a 2×TY medium (1.6% Bacto trypton, 1% Yeast extract, 0.5%NaCl), collected at an OD₆₆₀ of about 0.3, washed with a TM bufferdescribed below, then suspended in an NTG solution (prepared bydissolving NTG at a concentration of 0.2 mg/ml in TM buffer), andtreated at 37° C. for 0-90 minutes. The cell was washed with TM bufferand 2×TY medium, and then mutation was fixed by cultivation in 2×TYmedium overnight. Subsequently plasmid DNA was extracted from the cell,and introduced into an E. coli GT3 strain. Screening of candidatestrains was performed in the same manner as in the in vitro mutation,and mutants of lysine resistance (Lys^(R)) and AEC resistance (AEC^(R))were obtained.

    ______________________________________                                        (TM buffer)                                                                     Tris 50 mM                                                                    Maleic acid 50 mM                                                             (NH.sub.4).sub.2 SO.sub.4 1 g/L                                               MgSO.sub.4 ·7H.sub.2 O 0.1 g/L                                       Ca(NO.sub.3)2 5 mg/L                                                          FeSO.sub.4 ·7H.sub.2 O 0.25 mg/L                                   ______________________________________                                         pH was adjusted to 6.0 with NaOH.                                        

Total 180 strains of candidate strains obtained as described above(hydroxylamine treatment: 48 strains, NTG treatment: 132 strains) werespotted on the selection medium again, and AEC and L-lysine resistanceswere confirmed to obtain 153 strains. Taking a notice of difference inamino acid accumulation pattern in the medium, these 153 strains weredivided into 14 groups, and the AK activity was measured after selectingrepresentative strains of each of the groups. There was no largedifference in AK activity between the mutant strains obtained by thehydroxylamine treatment and the mutant strains obtained by the NTGtreatment. Thus the following experiments were performed withoutdistinguishing them.

(2-2-3) Isolation of lysc* Gene and Investigation on lysc* Product

No. 24, No. 43, No. 48, No. 60, No. 80, No. 117, No. 126, No. 149, No.150, No. 156, No. 158, No. 167, No. 169 and No. 172 were selected asrepresentative strains of the aforementioned 14 groups. Mutant plasmidsderived from pLYSC1 were recovered from each of them, and designated aspLYSC1*24, pLYSC1*43, pLYSC1*48, pLYSC1*60, pLYSC1*80, pLYSC1*117,pLYSC1*126, pLYSC1*149, pLYSC1*150, pLYSC1*156, pLYSC1*158, pLYSC1*167,pLYSC1*169 and pLYSC1*172, respectively. An AK completely deficientstrain GT3 was transformed with them and the wild type pLYSC1. Acell-free extract was prepared from each of transformed strains, and theenzyme activity of AKIII was measured.

The cell-free extract (crude enzyme solution) was prepared as follows. Atransformed strain was cultivated in a 2×TY medium, and collected at anOD₆₆₀ of about 0.8. Cells were washed with 0.02 M KH₂ PO₄ (pH 6.75)-0.03M β-mercaptoethanol under a condition of 0° C., and the cells wereruptured by sonication (0° C., 100 W, 30 minutes×4). A ruptured cellsolution was centrifuged at 33 krpm for 1 hour under a condition of 0°C. to obtain a supernatant, to which ammonium sulfate was added to give80% saturation. After centrifugation, a pellet was dissolved in 0.02 MKH₂ PO₄ (pH 6.75)-0.03 M β-mercaptoethanol, and stored at 0° C.overnight.

The enzyme activity of AKIII was measured in accordance with a method ofStadtman et al. (Stadtman, E. R., Cohen, G. N., LeBras, G., andRobichon-Szulmajster, H., J. Biol. Chem., 236, 2033 (1961)). Namely, areaction solution having the following composition was incubated at 27°C. for 45 minutes, and an FeCl₃ solution (2.8 N HCl 0.4 ml +12% TCA 0.4ml +5% FeCl₃. 6H₂ O/0.1 N HCl 0.7 ml) was added to develop a color,which was centrifuged followed by measurement of absorbance of asupernatant at 540 nm. The activity was indicated by an amount ofhydroxamic acid generated per minute (1 U=1 μmol/min). The molarabsorption coefficient was 600. Potassium aspartate was removed from thereaction solution to be used as a blank.

(Composition of Reaction Solution)

    ______________________________________                                        Reaction mixture *1 0.3 ml                                                      Hydroxylamine solution *2 0.2 ml                                              0.1 M Potassium aspartate (pH 7.0) 0.1 ml                                     Enzyme solution                                                               Water (balance)                                                                total 1.0 ml                                                               ______________________________________                                         *1: 1 M TrisHCl (pH 8.1) 9 ml + 0.3 M MgSO.sub.4 0.5 ml + 0.2 M ATP (pH       7.0) 5 ml                                                                     *2: 8 M Hydroxylamine solution was neutralized just before use with KOH. 

Various concentrations of L-lysine were added to the enzyme reactionsolution for measurement of the enzyme activity of AK, and the degree ofinhibition by L-lysine was examined. Results are shown in FIG. 6 andTable 3. The wild type and Nos. 24, 43, 48, 60, 80, 117 and 126 areshown in FIG. 6A. Nos. 149, 150, 156, 158, 167, 169 and 172 are shown inFIG. 6B.

As shown in these results, the wild type AKIII strongly sufferedinhibition by L-lysine, which was inhibited by 50% at about 0.45 mM ofL-lysine, and inhibited by about 100% at 5 mM. On the contrary, themutant AKIII's obtained this time had various degrees ofdesensitization, however, inhibition by L-lysine was desensitized in allof 14 species. Especially in the case of Nos. 24, 80, 117, 169 and 172,inhibition was scarcely observed even at 100 mM of L-lysine, and theyhad 50%-inhibitory-concentrations which were not less than 200 times ascompared with that of the wild type. The specific activity per totalprotein, which might be affected by growth situations of cell andpreparation of samples, was equal to or more than that of the wild typein almost all cases, in which there was little problem of decrease inactivity due to the introduction of mutation (Table 3). According tothis fact, it was postulated that an active center of AKIII wasindependent from a regulatory site by L-lysine with each other. In Table3, the inhibition desensitization degree (%) refers to an AK activity inthe presence of 100 mM of L-lysine with respect to an AK activity in theabsence of L-lysine in the reaction solution. The heat stability (%)refers to a ratio of activity maintenance after a treatment at 55° C.for 1.5 hour.

                  TABLE 3                                                         ______________________________________                                                             Degree of                                                   Specific activity desensitization Heat stability                              (U/mg protein) of inhibition (%)                                                                             .sup.*1 (%).sup.*2                          ______________________________________                                        Wild type                                                                             0.0247       0            18                                            No. 117 0.0069 120 0                                                          No. 24 0.0218 100 30                                                          No. 80 0.0244 99 36                                                           No. 172 0.0189 97 0                                                           No. 169 0.0128 96 2                                                           No. 150 0.0062 77 25                                                          No. 126 0.0250 61 39                                                          No. 149 0.0256 59 9                                                           No. 167 0.0083 43 45                                                          No. 48 0.0228 38 42                                                           No. 60 0.0144 35 9                                                            No. 158 0.0224 22 42                                                          No. 156 0.0101 18 2                                                           No. 43 0.0212 17 0                                                          ______________________________________                                         *1: AK activity (%) in the presence of 100 mM of Llysine with respect to      AK activity in the absence of Llysine                                         *2: ratio of activity maintenance (%) after treatment at 55° C. fo     1.5 hour                                                                 

Subsequently, the heat stability of the mutant enzymes was examined.When it is intended that an enzyme is improved to increase its activity,it is important that a created enzyme is maintained stably in cells.Measurement in vitro has some problems because of the difference inintracellular and extracellular protease activities and the influence ofbuffers for in vitro storage of enzymes. However, for convenience, theheat stability of the mutant AKIII's was investigated in vitro as oneparameter.

Judging from results of investigation on the inactivation temperature ofAKIII under various conditions, the ratio of activity maintenance aftera treatment at 55° C. for 90 minutes was measured. As shown in Table 3,half the enzymes were rather more excellent than the wild type.Generally, a mutant enzyme is often unstable as compared with a wildtype. However, some of the mutant AKIII's obtained this time weresuperior to the wild type in stability, and many of them seemed to befairly useful in practical use for L-lysine production.

(2-2-4) Determination of Base Sequence of Wild Type lysC and Mutant lysC

A nucleotide sequence of the wild type lysC gene obtained this time wasdetermined in accordance with an ordinary method by using a DNAsequencer ABI Model 373A (produced by Applied Biosystems Inc.) (SEQ IDNO:7). As a result, differences were found in six sites (two places atthe amino acid level) from an already published sequence of lysc of anE. coli K-12 JC411 strain (Cassan, M., Rarsot, C., Cohen, G. N., andPatte, J. C., J. Biol. Chem., 261, 1052 (1986)). It is speculated thatthe difference in six sites is due to the difference in bacterial strainused.

In the same manner, base sequences were determined for each of lysC*'sexisting on the 14 species of mutant pLYSC1's, and mutation points wereclarified. Results are shown in Table 4. In this table, indications inparentheses show mutations of amino acid residues based on mutations ofnucleotides. Types of mutations were 12 kinds because two sets (No. 4and No. 167, No. 24 and No. 80) had exactly the same mutation typesamong the 14 species. With respect to mutation types, Nos. 149, 150,156, 158, 167, 169 and 172 were obtained by the hydroxylamine treatment,and Nos. 24, 43, 48, 60, 80, 117 and 126 were obtained by the NTGtreatment. However, as for the pattern of mutation, any of them residedin mutation from cytosine to thymine, or mutation from guanine toadenine on a coding strand due to mutation from cytosine to thymine on anoncoding strand.

                  TABLE 4                                                         ______________________________________                                        Determination of mutation points of lysC*                                                                Mutation point                                       lysC* mutation type Mutagen (amino acid change)                             ______________________________________                                        No. 126     N          GGT→GA*T (.sup.323 Gly→Asp)                No. 43 N GGT→GA*T (.sup.323 Gly→Asp)                              GGC→GA*C (.sup.408 Gly→Asp)                                   No. 149 H CGT→T*GT ( .sup.34 Arg→Cys)                             GGT→GA*T (.sup.323 Gly→Asp)                                   No. 48/167 N/H CTC→T*TC (.sup.325 Leu→Phe)                      No. 150 H ATG→ATA* (.sup.318 Met→Ile)                           No. 172 H .sup.775 C→T (silent)                                          ATG→ATA* (.sup.318 Met→Ile)                                     GTG→A*TG (.sup.349 Val→Met)                                   No. 117 N TCA→TT*A (.sup.345 Ser→Leu)                           No. 158 H GTG→A*TG (.sup.347 Val→Met)                           No. 24/80 N/N ACC→AT*C (.sup.352 Thr→Ile)                       No. 169 H .sup.923 C→T (silent)                                          ACC→AT*C (.sup.352 Thr→Ile)                                     TCT→TT*T (.sup.369 Ser→Phe)                                   No. 60 N .sup.859 G→A (silent)                                           GAA→A*AA (.sup.164 Glu→Lys)                                   No. 156 H ATG→ATA* (.sup.417 Met→Ile)                             TGT→TA*T (.sup.419 Cys→Tyr)                                     .sup.2014 C→T (silent)                                             ______________________________________                                         *: H; hydroxylamine treatment, N; NTG treatment                          

EXAMPLE 3

Fermentation Production of L-lysine with Strain Being Introduced daDA*

In order to produce L-lysine by using E. coli, as indicated in JapanesePatent Application Laid-open No. 56-18596, U.S. Pat. No. 4,346,170 andApplied Microbiology and Biotechnology, 15, 227-231 (1982), it isconsidered to be essential that a host for enhancing DDPS has anaspartokinase which is changed not to suffer inhibition by L-lysine.L-threonine-producing bacteria may be exemplified as such a strain. Asfor L-threonine-producing E. coli, a B-3996 strain has the highestproductivity among those known at present. Thus the B-3996 strain wasused as a host for evaluating dapA*. The B-3996 strain harbors pVIC40extrachromosomally as a sole plasmid. Details are described in JapanesePatent Laid-open No. 3-501682 (PCT). This microorganism is deposited inResearch Institute for Genetics and Industrial Microorganism Breedingunder a registration No. of RIA 1867.

On the other hand, dapA* contained in pdapAS24 (in which the 118thhistidine residue replaced with a tyrosine residue) was selected asdapA* to be introduced into E. coli, judging from the degree ofdesensitization of inhibition and the specific activity of the enzyme.At first, in order to increase the expression amount of dapA* andincrease stability of the plasmid, mutant dapA* having existed onpdapAS24 (hereinafter referred to as "dapA*24") was ligated at thedownstream from a promoter of a tetracycline resistance gene of pVIC40,and RSF24P was obtained as shown in FIG. 7.

A strain obtained by introducing the plasmid RSF24P into an E. coliJM109 strain was designated as AJ12395, which is deposited in NationalInstitute of Bioscience and Human Technology of Agency of IndustrialScience and Technology on October 28, 1993, as accession number of FERMP-13935, and transferred from the original deposition to internationaldeposition based on Budapest Treaty on Nov. 1, 1994, and has beendeposited as accession number of FERM BP-4858. Strains harboring pdapAS8and pdapAS9 were not deposited. However, all of the mutation points ofdapA* on each of the plasmids have been clarified as described above.Thus it is easy for those skilled in the art that the plasmid isrecovered from the aforementioned deposited bacterium by using a methodof Maniatis et al. (Sambrook, J., Fritsch, E. F., Maniatis, T.,Molecular Cloning, Cold Spring Harbor Laboratory Press, 1.21 (1989)),and a target gene is obtained by using a site-directed mutagenesismethod (Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning,Cold Spring Harbor Laboratory Press, 15.63 (1989)).

pVIC40 was deleted from the B-3996 strain in accordance with an ordinarymethod, and a B-399 strain was obtained as a strain having no plasmid.The plasmid RSF24P was introduced into the B-399 strain in accordancewith an ordinary method, and B-399/RSF24P was obtained. The L-lysineproductivity of B-399/RSF24P was evaluated.

On the other hand, RSFP was constructed as a control plasmid. Namely, alarge fragment was selected from digest of pVIC40 double-degested withBamHI and DraI as shown in FIG. 7, and it was blunt-ended with DNApolymerase Klenow fragment. The blunt-ended large fragment wasself-ligated to obtain the plasmid RSFP. RSFP was introduced into theB-399 strain in accordance with an ordinary method, and B-399/RSFP wasobtained. The L-lysine productivity was also evaluated for B-399/RSFP.

The cultivation was performed at an agitation of 114-116 rpm under acondition of a cultivation period of 48 hours and a temperature of 37°C. by using the following medium. Results are shown in Table 5.

(Medium for L-lysine Production)

    ______________________________________                                        A:   (NH.sub.4).sub.2 SO.sub.4                                                                               16 g/L                                            KH.sub.2 PO.sub.4   1 g/L                                                     MgSO.sub.4 ·7H.sub.2 O   1 g/L                                       FeSO.sub.4 ·7H.sub.2 O 0.01 g/L                                      MnSO.sub.4 ·5H.sub.2 O 0.01 g/L                                      Yeast Ext. (Difco)   2 g/L                                                    L-methionine  0.5 g/L                                                         L-threonine  0.1 g/L                                                          L-isoleucine 0.05 g/L                                                         pH is adjusted to 7.0 with KOH to be (16/20 volume)                           autoclave at 115° C. for 10 minutes.                                  B: 20% Glucose (autoclave at 115° C. for 10  (4/20 volume)                                          minutes)                                         C: Pharmacopoeial CaCO.sub.3 (heat-sterilized in dry (30 g/L)                  state at 180° C. for 2 days)                                        ______________________________________                                    

A and B are mixed in the ratio of A:B=4:1, 30 g of C is added to 1 L ofthe mixture and dissolved, and antibiotics (streptomycin: 100 μg/ml,kanamycin: 5 μg/ml) are added.

                  TABLE 5                                                         ______________________________________                                                      Production amount of                                              Bacterial strain L-lysine hydrochloride                                     ______________________________________                                        B-399/RSF24P  4.1 g/L                                                           B-399/RSFP   0 g/L                                                          ______________________________________                                    

EXAMPLE 4

Fermentation Production of L-lysine with Strain Being Introduced dapA*and lysC* (I)

The effect of the mutant DDPS on L-lysine production has been shown inExample 3. In order to achieve further improvement, the mutant AKIIIgene obtained in Example 2 was allowed to co-exist with the mutant DDPSgene. The mutant AKIII gene to co-exist with the mutant DDPS gene wasselected as originating from the No. 80 strain (lysC*80), judging fromthe enzyme activity, heat stability and the like.

lysC*80 was used after excising it from a plasmid pLLC*80 (FIG. 8)prepared by alternatively ligating lysC* having existed on pLYSC1*80(hereinafter referred to as "lysC*80") at the downstream of a lacZpromoter of vector pHSG399 (produced by Takara Shuzo Co., Ltd.) whichhas an inverted-directional-insertion site with respect to pUC18 inorder to increase the expression amount of lysC*. pLLC*80 is a plasmidprepared to arrange lysC*80 to allow the direction of transcription tohave a normal orientation with respect to the lacZ promoter in order toimprove the productivity of L-lysine because lysC*80 on pLYSC1*80 hasits direction of transcription arranged in a reversed orientation withrespect to the lacZ promoter.

A plasmid, RSFD80, having dapA* and lysC* was prepared from pLLC*80 andRSF24P obtained in Example 3 as shown in FIG. 9. RSFD80 includes dapA*24and lysC*80 arranged in this order to allow the direction oftranscription to have a normal orientation with respect to tetP at thedownstream from a promoter (tetp) of a tetracycline resistance gene.

The RSFD80 μplasmid was introduced into an E. coli JM109 strain, whichwas designated as AJ12396. AJ12396 is deposited in National Institute ofBioscience and Human Technology of Agency of Industrial Science andTechnology on Oct. 28, 1993, as accession number of FERM P-13936, andtransferred from the original deposition to international depositionbased on Budapest Treaty on Nov. 1, 1994, and has been deposited asaccession number of FERM BP-4859. Strains harboring pLYSC1*24,pLYSC1*43, pLYSC1*48, pLYSC1*60, pLYSC1*117, pLYSC1*126, pLYSC1*149,pLYSC1*150, pLYSC1*156, pLYSC1*158, pLYSC1*167, pLYSC1*169 andpLYSC1*172 were not deposited. However, all of the mutation points oflysC* on each of the plasmids have been clarified as described above.Thus it is easy for those skilled in the art that the plasmid isrecovered from the aforementioned deposited bacterium by using a methodof Maniatis et al. (Sambrook, J., Fritsch, E. F., Maniatis, T.,Molecular Cloning, Cold Spring Harbor Laboratory Press, 1.21 (1989)),and a target gene is obtained by using a site-directed mutagenesismethod (Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning,Cold Spring Harbor Laboratory Press, 15.63 (1989)). RSFD80 wasintroduced into B-399 strain in accordance with an ordinary method, andB-399/RSFD80 was obtained. The L-lysine productivity of B-399/RSFD80 wasevaluated. The L-lysine productivity was also evaluated for B-399/RSFPas a control.

The cultivation was performed at an agitation of 114-116 rpm under acondition of a cultivation period of 48 hours and a temperature of 37°C. by using the same medium for production of L-lysine as in Example 3.Results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                      Production amount of                                              Bacterial strain L-lysine hydrochloride                                     ______________________________________                                        B-399/RSFD80  9.2 g/L                                                           B-399/RSFP   0 g/L                                                          ______________________________________                                    

EXAMPLE 5

Fermentation Production of L-lysine with Strain Being Introduced dapA*and lysC* (II)

It has been confirmed in Example 4 that the productivity of L-lysine canbe improved by allowing the bacterium belonging to the genus Escherichiato harbor the mutant dapA gene and the mutant lysC gene. Experimentswere performed to confirm whether or not this effect was maintained whenthe host is changed.

An E. coli W3110(tyrA) strain was used as a host. The W3110(tyrA) strainis described in detail in European Patent Publication No. 488424/92. Itspreparation method will be briefly described as follows. The E. coliW3110 strain was obtained from National Institute of Genetics(Mishima-shi, Shizuoka-ken, Japan). This strain was spread on an LBplate containing streptomycin, and a streptomycin resistant strain wasobtained by selecting strains which formed colonies. The selectedstreptomycin resistant strain was mixed with an E. coli K-12 ME8424strain, and stationarily cultivated in a complete medium (L-Broth: 1%Bacto trypton, 0.5% Yeast extract, 0.5% NaCl) under a condition of 37°C. for 15 minutes to induce conjugation. The E. coli K-12 ME8424 strainhas genetic characters of (HfrPO₄₅, thi, relA1, tyrA::Tn10, ung-1,nadB), which is available from National Institute of Genetics.

The culture was then spread on a complete medium (L-Broth: 1% Bactotrypton, 0.5% Yeast extract, 0.5% NaCl, 1.5% agar) containingstreptomycin, tetracycline and L-tyrosine, and a colony-forming strainwas selected. This strain was designated as E. coli W3110(tyrA) strain.

By the way, European Patent Publication No. 488424/92 describes manystrains formed by introducing plasmids into the W3110(tyrA) strain. Forexample, a strain obtained by introducing a plasmid pHATerm isdesignated as E. coli W3110(tyrA)/pHATerm strain, and deposited inNational Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology, to which a registration No. of FERMBP-3653 is given. The W3110(tyrA) strain can be also obtained by curingthe plasmid pHATerm from the E. coli W3110(tyrA)/pHATerm strain. Thecuring of the plasmid can be performed in accordance with an ordinarymethod.

The plasmid RSFD80 containing both of dapA* and lysc* obtained inExample 4 was introduced into the W3110(tyrA) obtained as describedabove, and W3110(tyrA)/RSFD80 was obtained. The L-lysine productivitywas evaluated for W3110(tyrA)/RSFD80. As a control, RSFP was introducedinto the W3110(tyrA) strain in accordance with an ordinary method, andW3110(tyrA)/RSFP was obtained. The L-lysine productivity was alsoevaluated for W3110(tyrA)/RSFP as a control.

The cultivation was performed at an agitation of 114-116 rpm under acondition of a cultivation period of 48 hours and a temperature of 37°C. by using the aforementioned medium for L-lysine production. Resultsare shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                        Production amount of                                            Bacterial strain L-lysine hydrochloride                                     ______________________________________                                        W3110 (tyrA) /RSFD80                                                                          8.9 g/L                                                         W3110 (tyrA) /RSFP   0 g/L                                                  ______________________________________                                    

EXAMPLE 6

Analysis of Rate Determining Steps of L-lysine Biosynthesis System andImprovement in L-lysine Productivity of L-lysine-producing BacteriaBelonging to the Genus Escherichia

It was tried to improve the L-lysine productivity by analyzing ratedetermining steps of the L-lysine biosynthesis system of E. coli andenhancing genes for enzymes which catalyze the steps.

<1> Identification of the First Rate Determining Steps (6-1-1)Preparation of Genes of L-lysine Biosynthesis System

The rate determining step was identified by isolating various genes ofthe L-lysine biosynthesis system, introducing these genes into E. coli,and examining effects of each of the genes on the L-lysine productivity.The introduced genes for enzymes of the L-lysine biosynthesis system,and the enzymes encoded by them are as follows.

ppc: phosphoenolpyruvate carboxylase

aspc: aspartate aminotransferase

lysC: aspartokinase III

lysC*80: inhibition-desensitized aspartokinase III

asd: aspartate semialdehyde dehydrogenase

dapA: dihydrodipicolinate synthase

dapA*24: inhibition-desensitized dihydrodipicolinate synthase

dapB: dihydrodipicolinate reductase

DDH: diaminopimelate dehydrogenase (originating from Brevibacteriumlactofermentum)

lysA: diaminopimelate decarboxylase

The L-lysine biosynthesis system from phosphoenolpyruvic acid toL-lysine can be thoroughly covered by the genes described above. Thedapc, dapD, dapE and dapF genes, among the genes of the L-lysinebiosynthesis system originally possessed by E. coli, are replaced withthe gene DDH coding for DDH (diaminopimelate dehydrogenase) ofBrevibacterium lactofermentum which can catalyze reactions concerningthese gene products by itself. The W3110(tyrA) strain of the E. coliK-12 series was used as a host for introducing these genes.

The dapA and dapA*24 genes were respectively obtained by excision frompdapA2 and pdapAS24 (see Example 1) with EcoRI and KpnI (FIG. 10). Thesegenes were ligated with pMW118 which was digested with EcoRI and KpnI toobtain pdapA and pdapA*. The lysC and lysC*80 genes were respectivelyobtained by excision from pLYSC1 and pLLC*80 (see Example 2) with EcoRIand SphI. These genes were ligated with pMW119 which was digested withEcoRI and SphI to obtain plysc and plysC* (FIG. 11).

The ppc gene was obtained from a plasmid pT2 having this gene. pT2 wascut with SmaI and ScaI, and the termini were blunt-ended, followed byinsertion into a SmaI site of pMW118 to obtain a plasmid pppc (FIG. 12).E. coli F15 (AJ12873) harboring pT2 is deposited in National Instituteof Bioscience and Human Technology of Agency of Industrial Science andTechnology under an accession number of FERM BP-4732.

The aspC gene was obtained from a plasmid pLF4 (Inokuchi, K. et al.,Nucleic Acids Res., 10, 6957 (1982)) having this gene (FIG. 13). pLF4was cut with PvuII and StuI, and the termini were blunt-ended, followedby insertion into a SmaI site of pMW119 to obtain a plasmid paspc.

The asd gene was obtained from a plasmid pAD20 (Haziza, C. et al., EMBO,1, 379 (1982)) having this gene. pAD20 was cut with AseI and ClaI, andthe termini were blunt-ended, followed by insertion into a SmaI site ofpMW118 to obtain a plasmid pasd (FIG. 14).

The dapB gene was obtained by amplifying a dapB gene from chromosomalDNA of an E. coli W3110 strain by means of the PCR method by using twospecies of oligonucleotide primers (SEQ ID NO:9, NO:10) prepared on thebasis of a nucleotide sequence of a known dapB gene (Bouvier, J. et al.,J. Biol. Chem., 259, 14829 (1984)) (FIG. 15). An obtained amplified DNAfragment was cut with AseI and DraI, and the termini were blunt-ended,followed by insertion into a SmaI site of pMW119 to obtain a plasmidpdapB.

The DDH gene was obtained by amplifying a DDH gene from chromosomal DNAof Brevibacterium lactofermentum ATCC13869 by means of the PCR method byusing two species of oligonucleotide primers (SEQ ID NO:11, NO:12)prepared on the basis of a known nucleotide sequence of a DDH gene ofCorynebacterium glutamicum (Ishino, S. et al., Nucleic Acids Res., 15,3917 (1987)). An obtained amplified DNA fragment was cut with EcoT22Iand AvaI, and the termini were blunt-ended, followed by insertion into aSmaI site of pMW119 to obtain a plasmid pDDH (FIG. 16).

The lysA gene was obtained by amplifying a lysA gene from chromosomalDNA of an E. coli W3110 strain by means of the PCR method by using twospecies of oligonucleotide primers (SEQ ID NO:13, NO:14) prepared on thebasis of a nucleotide sequence of a known lysA gene (Stragier, P. etal., J. Mol. Biol., 168, 321 (1983)). An obtained amplified DNA fragmentwas cut with SplI and BclI, and the termini were blunt-ended, followedby insertion into a SmaI site of pMW118 to obtain a plasmid plysA (FIG.17).

Confirmation of the fact that each of the aforementioned genes wascloned was performed by cutting them with restriction enzymes shown inthe figures. The vectors pMW118 and pMW119 (produced by Nippon Gene)used for cloning of these genes were selected because they were able toco-exist in cells of E. coli together with RSF1010 as a vector used forpreparation of plasmids for lysine production described below, and alsohad a stable distribution mechanism.

(6-1-2) L-lysine Productivity of E. coli with Introduced Genes ofL-lysine Biosynthesis System

E. coli W3110(tyrA) was transformed with each of the plasmids containingthe genes of the L-lysine biosynthesis system described above, andobtained transformants were cultivated to perform L-lysine production.The cultivation was performed for 30 hours under a condition of acultivation temperature of 37° C. and an agitation of 114-116 rpm byusing the following medium. Results are shown in Table 8.

(Medium Composition)

    ______________________________________                                        Glucose                40     g/l                                               MgSO.sub.4 •7H.sub.2 O 1 g/l                                            (NH.sub.4).sub.2 SO.sub.4 16 g/l                                              KH.sub.2 PO.sub.4 1 g/l                                                       FeSO.sub.4 •7H.sub.2 O 0.01 g/l                                         NnSO.sub.4 •5H.sub.2 O 0.01 g/l                                         Yeast Ext. (Difco) 2 g/l                                                      L-tyrosine 0.1 g/l                                                          pH was adjusted to 7.0 with KOH to be                                           autoclaved at 115° C. for 10 minutes (Glucose                          and MgSO.sub.4.7H.sub.2 O were separately sterilized).                             Pharmacopoeial CaCO.sub.3                                                                         25     g/l                                         (heat-sterilized in dry state at 180° C. for 2                           days)                                                                         Antibiotics                                                                   (streptomycin 20 mg/l or ampicillin 50 mg/l                                   depending on species of plasmids to be                                        introduced)                                                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                                       Production amount of                                              L-lysine hydrochloride Yield versus                                          Bacterial strain (g/l) sugar (%)                                            ______________________________________                                        W3110(tyrA)    0.08          0.2                                                W3110(tyrA)/pppc 0.08 0.2                                                     W3110(tyrA)/paspC 0.12 0.3                                                    W3110(tyrA)/plysC 0.08 0.2                                                    W3110(tyrA)/plysC* 2.27 5.57                                                  W3110(tyrA)/pasd 0.12 0.3                                                     W3110(tyrA)/pdapA 2.32 5.70                                                   W3110(tyrA)/pdapA* 3.63 8.90                                                  W3110(tyrA)/pdapB 0.08 0.2                                                    W3110(tyrA)/pDDH 0.08 0.2                                                     W3110(tyrA)/plysA 0.12 0.3                                                  ______________________________________                                    

i E. coli W3110(tyrA) became to produce L-lysine by introduction ofplysC*, pdapA or pdapA*. Since both of lysC product and dapA productsuffer feedback inhibition by L-lysine, it can be postulated that theseenzymes are major regulatory points in L-lysine biosynthesis. Thereaction catalyzed by dapA product exists in a position of branching toa biosynthesis system for L-threonine, L-methionine and L-isoleucine anda biosynthesis system for L-lysine, and is the first step of thebiosynthesis system inherent to L-lysine. It was already reported thatE. coli also becomes to produce L-lysine by amplification of a wild typedapA (Eur. J. Appl. Microbiol. Biotechnol., 15, 227 (1982)), which hasbeen also confirmed from the result described above. On the other hand,the result of Example 3 has been confirmed again in that the yield ofL-lysine is further increased when dapA* as an inhibition-desensitizedtype gene is introduced into E. coli.

Crude enzyme solutions were prepared from W3110(tyrA), W3110(tyrA)/pdapAand W3110(tyrA)/pdapA* in the same manner as in Example 1, the DDPS(dihydrodipicolinate synthase) activity was measured, and the degree ofinhibition of the DDPS activity by L-lysine was examined. Results areshown in Table 9.

                  TABLE 9                                                         ______________________________________                                                                 Degree of                                               Specific desensitization of                                                  Bacterial strain activity *1 inhibition *2                                  ______________________________________                                        W3110(tyrA)     0.0423   50                                                     W3110(tyrA)/pdapA 0.2754 22.9                                                 W3110(tyrA)/pdapA* 0.1440 76.5                                              ______________________________________                                         *1: μmols/min/mg protein                                                   *2: ratio of activity maintenance (%) in the presence of 5 mM of Llysine 

The inhibition-desensitized dapA* product probably has a large effect onL-lysine production because it has a high degree of desensitization ofinhibition although it has a lower specific activity than the wild typeenzyme (about 1/2). The necessity of the desensitization of inhibitionof the dapA product has been shown for L-lysine production.

In addition, the fact that lysc* has an effect on L-lysine productioncan be considered as follows. The first rate determining step is a stepat which HD (homoserine dehydrogenase: product of thrA or metLM)competes with DDPS (dapA product) in acquiring ASA(aspartate-β-semialdehyde) as a substrate to serve at a branching pointof the biosynthesis system, and when dapA is enhanced as describedabove, the reaction flows in a direction of L-lysine biosynthesis. Onthe other hand, it is speculated that when the supply amount of ASA isincreased by enhancing lysc which participates in a reaction furtherupstream from dapA, any of reactions relevant to HD and DDPS is alsofacilitated, and thus the production amount of L-lysine has been alsoincreased. However, this effect is scarcely obtained by enhancement ofthe wild type lysC only. This is probably because the inhibition of thewild type AKIII (lysC product) by L-lysine is more strict than that ofthe wild type DDPS (AKIII and DDPS are inhibited by about 100% and 80%respectively in the presence of 5 mM of L-lysine).

According to the facts described above, it was judged that the reactionby DDPS having a higher lysine-producing effect was the first ratedetermining step, and it was postulated that the reaction by AKIII wasthe second rate determining step.

<2> Identification of the Second Rate Determining Step

The second rate determining step was identified by enhancing variousgenes of the L-lysine biosynthesis system in strains with introduceddapA*. In order that other plasmids were stably harbored when they wereintroduced into E. coli harboring a plasmid containing dapA*, dapA* wastransferred from pdapA to RSF1010, and RSF24P was obtained (FIG. 7). E.coli W3110(tyrA) was transformed with the plasmid RSF24P having dapA*.

Plasmids having genes of the L-lysine biosynthesis system wereintroduced into E. coli W3110(tyrA)/RSF24P. Two species of plasmids,namely RSF24P and a plasmid containing another gene of the L-lysinebiosynthesis system, co-exist in each of obtained transformants. TheL-lysine productivity was examined for these strains in the same manneras in (6-1-2). Results are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                                        Production amount                                                of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        W3110(tyrA)/RSF24P                                                                            3.63          8.9                                               W3110(tyrA)/RSF24P + pppc 3.67 9.0                                            W3110(tyrA)/RSF24P + paspC 3.59 8.8                                           W3110(tyrA)/RSF24P + plysC 3.42 8.4                                           W3110(tyrA)/RSF24P + plysC* 9.17 22.5                                         W3110(tyrA)/RSF24P + pasd 3.75 9.2                                            W3110(tyrA)/RSF24P + pdapA 3.55 8.7                                           W3110(tyrA)/RSF24P + pdapA* 3.46 8.5                                          W3110(tyrA)/RSF24P + pdapB 4.08 10.0                                          W3110(tyrA)/RSF24P + pDDH 3.67 9.0                                            W3110(tyrA)/RSF24P + plysA 3.55 8.7                                         ______________________________________                                    

As a result, a remarkable enhancing effect on the L-lysine productivitywas found only in lysC*. The wild type lysC had no effect at all. Thisis probably because the inhibition by L-lysine is strong as describedabove. Thus it was confirmed that the reaction participated by lysC* wasthe second rate determining step.

lysc* was integrated into RSF24P, and RSFD80 was obtained (FIG. 9). Inthe same manner, lysC was integrated into RSF24P, and an obtainedplasmid was designated as RSFD1. These plasmids were introduced into E.coli W3110(tyrA), crude enzyme solutions were prepared, and the AKactivity and the degree of inhibition of AK activity by L-lysine wereexamined in the same manner as in (6-1-2). Results are shown in Table11.

                  TABLE 11                                                        ______________________________________                                                                Degree of                                               Bacterial strain Specific desensitization                                     for AK activity activity *1 of inhibition *2                                ______________________________________                                        W3110(tyrA)/RSF24P                                                                           0.94     42.9                                                    W3110(tyrA)/RSFD1 18.55 7.2                                                   W3110(tyrA)/RSFD80 33.36 98.8                                               ______________________________________                                         *1: nmols/min/mg protein                                                      *2: ratio of activity maintenance (%) in the presence of 5 mM of Llysine 

The specific activities of AK of the strains harboring the plasmids wereincreased 20-30 times by integrating lysC and lysC* into RSF24P. E. colihas three species of AK's, and lysC codes for AKIII among them. However,a total activity of the three species of AK's was measured in theexperiment described above. It is speculated that the inhibition byL-lysine also becomes high in the strain harboring RSFD1 with theinserted wild type lysc because the ratio occupied by AKIII is higherthan those by AKI and AKIII as compared with the control(W3110(tyrA)/RSF24P), resulting in no indication of the effect onenhancement of the L-lysine productivity. On the other hand, it wasrevealed that the inhibition was desensitized for about 100% of AKIII inthe strain harboring RSFD80, and this fact contributed to theimprovement in L-lysine production.

<3> Identification of the Third Rate Determining Step

Next, various plasmids of the L-lysine biosynthesis system wereintroduced into E. coli W3110(tyrA)/RSFD80, and cultivation for L-lysineproduction was performed. Cultivation results are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                                         Production amount                                               of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        W3110(tyrA)/RSFD80                                                                             9.17          22.5                                             W3110(tyrA)/RSFD80 + pppc 8.97 22.0                                           W3110(tyrA)/RSFD80 + paspC 9.05 22.2                                          W3110(tyrA)/RSFD80 + plysC 8.56 21.0                                          W3110(tyrA)/RSFD80 + plysC* 8.15 20.0                                         W3110(tyrA)/RSFD80 + pasd 8.35 20.5                                           W3110(tyrA)/RSFD80 + pdapA 8.56 21.0                                          W3110(tyrA)/RSFD80 + pdapA* 8.15 20.0                                         W3110(tyrA)/RSFD80 + pdapB 10.80 26.5                                         W3110(tyrA)/RSFD80 + pDDH 8.56 21.0                                           W3110(tyrA)/RSFD80 + plysA 8.48 20.8                                        ______________________________________                                    

An enhancing effect on the L-lysine productivity was observed only indapB, and it was found that the reaction participated by dapB was thethird rate determining step. Thus dapB was inserted into RSFD80, andpCAB1 was obtained (FIG. 18). This plasmid was introduced into E. coliW3110(tyrA), a crude enzyme solution was prepared, and the enzymeactivity of DDPR (dihydrodipicolinate reductase) was measured inaccordance with a method described by Tamir, H. and Gilvarg, C., J.Biol. Chem., 249, 3034 (1974). In the same manner, crude enzymesolutions were prepared from a strain harboring only RSFD80 and a strainharboring both RSFD80 and pdapB, and the DDPR activity was measured.Results are shown in Table 13.

                  TABLE 13                                                        ______________________________________                                                          Specific activity                                             Bacterial strain (μmols/min/mg protein)                                  ______________________________________                                        W3110(tyrA)/RSFD80                                                                              0.027                                                         W3110(tyrA)/RSFD80 + pdapB 0.092                                              W3110(tyrA)/PCAB1 0.178                                                     ______________________________________                                    

The DDPR activity was increased about 3 times in the strain harboringRSFD80 and pdapB, and it was increased about 6.5 times in the strainharboring pCAB1 in which dapB was inserted into RSFD80, as compared withthe control (strain harboring RSFD80 only). According to the fact thatboth W3110(tyrA)/RSFD80+pdapB and W3110(tyrA)/pCAB1 had equivalentL-lysine accumulation of 10.8 g/l, it was judged that dapB was providedin a sufficient amount for L-lysine production, and the rate determiningstep was shifted to the next step.

<4> Identification of the Fourth Rate Determining Step

Next, the fourth rate determining step was identified by using theplasmid pCAB1 harboring lysc*, dapA* and dapB. Various plasmids of theL-lysine biosynthesis system were introduced into E. coliW3110(tyrA)/pCAB1, and cultivation for L-lysine production wasperformed. Cultivation results are shown in Table 14.

                  TABLE 14                                                        ______________________________________                                                        Production amount                                                of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        W3110(tyrA)/pCAB1                                                                             10.80         26.5                                              W3110(tyrA)/pCAB1 + pppc 11.00 27.0                                           W3110(tyrA)/pCAB1 + paspC 10.88 26.7                                          W3110(tyrA)/pCAB1 + plysC 10.60 26.0                                          W3110(tyrA)/pCAB1 + plysC* 10.39 25.5                                         W3110(tyrA)/pCAB1 + pasd 10.19 25.0                                           W3110(tyrA)/pCAB1 + pdapA 10.72 26.3                                          W3110(tyrA)/pCAB1 + pdapA* 10.80 26.5                                         W3110(tyrA)/pCAB1 + pdapB 10.92 26.8                                          W3110(tyrA)/pCAB1 + pDDH 12.23 30.0                                           W3110(tyrA)/pCAB1 + plysA 10.60 26.0                                        ______________________________________                                    

An enhancing effect on the L-lysine productivity was observed only inDDH, and it was found that the reaction catalyzed by DDH was the fourthrate determining step. In addition, SDAP (N-succinyl-L,L-α,ε-diaminopimelic acid) detected in a culture broth of the DDHnon-introduced strain was not detected in a culture broth of the DDHintroduced strain. Detection of SDAP was performed by means of TLCdevelopment (composition of development solvent; methanol:water:10NHCl:pyridine =80:17.5:2.5:10) (Bouvier, J., Richaud, C., Higgins, W.,Bogler, O. and Stragier, P., J. Bacteriol., 174, 5265 (1992)). Further,the color of broth was brown in the case of the DDH non-introducedstrain, but it was changed to yellow in the case of the DDH introducedstrain. Thus DDH was integrated into pCAB1 to prepared a plasmid pCABD2(FIG. 19), and the DDH activity of E. coli W3110(tyrA) transformed withthis plasmid was measured. The DDH enzyme activity was measured inaccordance with a literature (Azizono, Haruo, Fermentation and Industry,45, 964 (1987)). Results are shown in Table 15.

                  TABLE 15                                                        ______________________________________                                                          Specific activity                                             Bacterial strain (μmols/min/mg protein)                                  ______________________________________                                        W3110(tyrA)/pCAB1 0.000                                                         W3110(tyrA)/pCAB1 + pDDH 0.799                                                W3110(tyrA)/pCABD2 2.214                                                    ______________________________________                                    

The DDH activity was not detected in the control (W3110(tyrA)/pCAB1)because DDH was originally not present in E. coli. The specific activityof DDH of the strain harboring pCABD2 (W3110(tyrA)/pCABD2) was about 2.5times that of the strain harboring pDDH (W3110(tyrA)/pCAB1+pDDH),however, the both strain had an equivalent L-lysine accumulation amount(12.23 g/l). Thus it was judged that the DDH expression amount of pCABD2was a sufficient amount.

<5> Analysis of Rate Determining Steps Among dapC, dapD, dapE and dapF

Next, in order to examine a rate limiting order of dapC, dapD, dapE anddapF replaced by DDH in the analysis described above, at first thesegenes were cloned. dapc was not cloned because of no report on its basesequence, however, the remaining three species of genes were cloned inaccordance with the PCR method.

The dapD gene was obtained by amplifying a dapD gene from chromosomalDNA of an E. coli W3110 strain by means of the PCR method by using twospecies of oligonucleotide primers (SEQ ID NO:15, NO:16) prepared on thebasis of a nucleotide sequence of a known dapD gene (Richaud, C. et al.,J. Biol. Chem., 259, 14824 (1984)). An obtained amplified DNA fragmentwas cut with Eco0109I and SacI, and the termini were blunt-ended,followed by insertion into a SmaI site of pMW118 to obtain a plasmidpdapD (FIG. 20).

The dapE gene was obtained by amplifying a dapE gene from chromosomalDNA of an E. coli W3110 strain by means of the PCR method by using twospecies of oligonucleotide primers (SEQ ID NO:17, NO:18) prepared on thebasis of a nucleotide sequence of a known dapE gene (Bouvier, J. et al.,J. Bacteriol., 174, 5265 (1992)). An obtained amplified DNA fragment wascut with MunI and BalII, and the termini were blunt-ended, followed byinsertion into a SmaI site of pMW118 to obtain a plasmid pdapE (FIG.21).

The dapF gene was obtained by amplifying a dapF gene from chromosomalDNA of an E. coli W3110 strain by means of the PCR method by using twospecies of oligonucleotide primers (SEQ ID NO:19, NO:20) prepared on thebasis of a nucleotide sequence of a known dapF gene (Richaud, C. et al.,Nucleic Acids Res., 16, 10367 (1988)). An obtained amplified DNAfragment was cut with PstI, and the termini were blunt-ended, followedby insertion into a SmaI site of pMW118 to obtain a plasmid pdapF (FIG.22).

Each of the plasmids obtained as described above was introduced intoW3110(tyrA)/pCAB1, and cultivation for L-lysine production wasperformed. In the previous experiment, the changes were observed in thecolor of broth and in the presence or absence of accumulation of theintermediate (SDAP) in addition to the L-lysine production amountbetween before and after the introduction of DDH. Thus the analysis ofthe rate determining step was performed also by using them as indexes.Results are shown in Table 16.

                  TABLE 16                                                        ______________________________________                                                        Production                                                       amount of   Accu-                                                             L-lysine Yield  mula-                                                         hydro- versus Color tion                                                      chloride sugar of of                                                         Bacterial strain (g/l) (%) broth SDAP                                       ______________________________________                                        W3110(tyrA)/pCAB1                                                                             10.80    26.5    brown +                                        W3110(tyrA)/pCAB1 + pdapD 11.08 27.2 yellow +                                 W3110(tyrA)/pCAB1 + pdapE 11.12 27.3 brown -                                  W3110(tyrA)/pCAB1 + pdapF 10.96 26.9 brown +                                  W3110(tyrA)/pCABD2 12.23 30.0 yellow -                                      ______________________________________                                    

The production amount of L-lysine was increased a little by theenhancement of dapD or dapE, but DDH was not exceeded. Further, it wasfound that the change in color of broth and the accumulation of SDAP asan intermediate observed upon the introduction of DDH were independentphenomena with each other, the change in color of broth resulted fromdapD, and the disappearance of SDAP resulted from dapE. The relationbetween dapE and SDAP may be postulated judging from the biosynthesispathway of L-lysine. The enhancement of dapF had no effect on theimprovement in L-lysine productivity.

dapE was excised from pdapE, and it was inserted into pdapD to prepare aplasmid pMWdapDE1 containing both dapE and dapD (FIG. 23). Further, afragment containing dapE and dapD was excised from pMWdapDE1, and it wasinserted into pCAB1 to prepare pCABDE1 (FIG. 24). Strains harboringpCAB1, pCABDE1 or pCABD2 and a strain harboring both pCABDE1 and pdapFwere prepared, and cultivation for L-lysine production was performed byusing these strains. Results are shown in FIG. 17.

                  TABLE 17                                                        ______________________________________                                                        Production                                                       amount of   Accu-                                                             L-lysine Yield  mula-                                                         hydro- versus Color tion                                                      chloride sugar of of                                                         Bacterial strain (g/l) (%) broth SDAP                                       ______________________________________                                        W3110(tyrA)/pCAB1                                                                             10.80    26.5    brown +                                        W3110(tyrA)/pCABDE1 12.23 30.0 yellow -                                       W3110(tyrA)/pCABDE1 11.82 29.0 yellow -                                       + pdapF                                                                       W3110(tyrA)/pCABD2 12.23 30.0 yellow -                                      ______________________________________                                    

It was found that the L-lysine production amount, the color of broth,and the presence or absence of accumulation of SDAP became equivalent tothose in the case of the production of DDH by enhancing dapD and dapE incombination. In addition, it was found that further enhancement of dapFhad no effect on the improvement in L-lysine productivity, and thereaction participated by dapF did not make rate limitation. The resultsdescribed above can be interpreted as follows.

Upon the step of introduction of pCAB1, intermediates are accumulated attwo steps of SKAP (N-succinyl-ε-keto-L-α-aminopimelic acid) and SDAP.Among these intermediates, SDAP was detected in an extracellular broth.Although SKAP was not detected, it was speculated to be accumulated inbacterial cells. The reason for such speculation resides in the color ofbroth. The color of broth is yellow in the case of the wild type strain(W3110(tyrA)) or the like producing no L-lysine. However, the brothbecomes brown probably due to bacteriolysis or the like when a load isapplied to growth. It is speculated that SDAP has a small load on growthbecause it is discharged to the outside of cells, and hence, the brothis improved to have a yellow color although the accumulation amount ofSDAP increases when SKAP is metabolized by the enhancement of only dapD.However, even if dapD is enhanced, the accumulation amount of L-lysinedoes not increase unless rate limitation by further downstream dapE isdesensitized.

<6> Conclusion

According to the results described above, it has been found that theL-lysine productivity is improved in a stepwise manner by performing (1)introduction of dapA*, (2) introduction of lysC*, (3) enhancement ofdapB, and (4) enhancement of DDH or dapD and dapE in bacteria belongingto the genus Escherichia. Further, E. coli, in which the L-lysineproductivity is improved in a stepwise manner, has been obtained.

<7> Analysis of Rate Determining Step of L-lysine Biosynthesis System inE. coli C Strain

In order to examine whether or not the conclusion obtained in theforegoing could be applied to strains other than the E. coli K-12series, rate determining steps of an L-lysine biosynthesis system of anE. coli C strain (IFO 13891) were analyzed in the same manner asdescribed above. The cultivation condition was the same as that of W3110(tyrA), however, L-tyrosine was not added to the medium.

(1) Identification of the First Rate Determining Step

The E. coli C strain (IFO 13891) transformed with plasmids containinggenes of the L-lysine biosynthesis system was cultivated in the mediumfor L-lysine production, and the production amount of L-lysinehydrochloride was measured. Results are shown in Table 18.

                  TABLE 18                                                        ______________________________________                                                      Production amount of                                                                        Yields                                               L-lysine hydrochloride versus                                                Bacterial strain (g/l) sugar (%)                                            ______________________________________                                        C             0.08          0.2                                                 C/pppc 0.08 0.2                                                               C/paspC 0.12 0.3                                                              C/plysC 0.08 0.2                                                              C/plysC* 0.12 0.3                                                             C/pasd 0.08 0.2                                                               C/pdapA 0.32 0.8                                                              C/pdapA* 0.71 1.75                                                            C/pdapB 0.12 0.3                                                              C/pDDH 0.08 0.2                                                               C/plysA 0.08 0.2                                                            ______________________________________                                    

In the same manner as in W3110 (tyrA), L-lysine was also accumulated inthe medium by the C strain by introducing the wild type dapA and furtherthe inhibition-desensitized type dapA*. lysC* had no effect on theL-lysine productivity.

(2) Identification of the Second Rate Determining Step p The plasmidRSF24P containing dapA* was introduced into the E. coli C strain, andplasmids containing genes of the L-lysine biosynthesis system werefurther introduced. Obtained transformants were cultivated in the mediumfor L-lysine production, and the production amount of L-lysinehydrochloride was measured. Results are shown in Table 19.

                  TABLE 19                                                        ______________________________________                                                       Production amount                                                 of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        C/RSF24P       0.71         1.75                                                C/RSF24P + pppc 0.71 1.74                                                     C/RSF24P + paspC 0.69 1.70                                                    C/RSF24P + plysC 0.65 1.60                                                    C/RSF24P + plysC* 1.82 4.50                                                   C/RSF24P + pasd 0.70 1.73                                                     C/RSF24P + pdapA 0.71 1.75                                                    C/RSF24P + pdapA* 0.69 1.70                                                   C/RSF24P + pdapB 0.99 2.45                                                    C/RSF24P + pDDH 0.73 1.80                                                     C/RSF24P + plysA 0.69 1.70                                                  ______________________________________                                    

It was found that lysC* had an effect on the improvement in L-lysineproductivity even in the case of the C strain with transformed dapA*,and the reaction participated by lysC* was the second rate determiningstep.

(3) Identification of the Third Rate Determining Step

The plasmid RSFD80 containing dapA* and lysC* was introduced into the E.coli C strain, and plasmids containing genes of the L-lysinebiosynthesis system were further introduced. Obtained transformants werecultivated in the medium for L-lysine production, and the productionamount of L-lysine hydrochloride was measured. Results are shown inTable 20.

                  TABLE 20                                                        ______________________________________                                                       Production amount                                                 of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        C/RSFD80       1.82         4.5                                                 C/RSFD80 + pppc 1.74 4.3                                                      C/RSFD80 + paspC 1.82 4.5                                                     C/RSFD80 + plysC 1.91 4.7                                                     C/RSFD80 + plysC* 1.74 4.3                                                    C/RSFD80 + pasd 1.82 4.5                                                      C/RSFD80 + pdapA 1.95 4.8                                                     C/RSFD80 + pdapA* 1.91 4.7                                                    C/RSFD80 + pdapB 2.31 5.7                                                     C/RSFD80 + pDDH 2.15 5.3                                                      C/RSFD80 + plysA 1.95 4.8                                                   ______________________________________                                    

In the same manner as in the W3110 strain, only dapB had an effect onthe improvement in L-lysine productivity, and it was found to be thethird rate determining step.

(4) Identification of the Fourth Rate Determining Step

The plasmid pCAB1 containing dapA*, lysC* and dapB was introduced intothe E. coli C strain, and plasmids containing genes of the L-lysinebiosynthesis system were further introduced. Obtained transformants werecultivated in the L-lysine-producing medium, and the production amountof L-lysine hydrochloride was measured. Results are shown in Table 21.

                  TABLE 21                                                        ______________________________________                                                       Production amount                                                 of L-lysine Yield versus                                                     Bacterial strain hydrochloride (g/l) sugar (%)                              ______________________________________                                        C/pCAB1        2.31         5.7                                                 C/pCAB1 + pppc 2.23 5.5                                                       C/pCAB1 + paspC 2.35 5.8                                                      C/pCAB1 + plysC 2.27 5.6                                                      C/pCAB1 + plysC* 2.19 5.4                                                     C/pCAB1 + pasd 2.23 5.5                                                       C/pCAB1 + pdapA 2.31 5.7                                                      C/pCAB1 + pdapA* 2.27 5.6                                                     C/pCAB1 + pdapB 2.23 5.5                                                      C/pCAB1 + pDDH 2.59 6.4                                                       C/pCAB1 + plysA 2.19 5.4                                                    ______________________________________                                    

In the same manner as in the W3110 strain, only DDH had an effect on theimprovement in L-lysine productivity, and it was found to be the fourthrate determining step.

(5) Analysis of Rate Determining Steps Among dapC, dapD, dapE and dapF

Plasmid harboring the dapD, dapE or dapF genes were introduced, insteadof DDH, into the E. coli C strain harboring pCAB1, and cultivation forL-lysine production was performed. Results are shown in Table 22.

                  TABLE 22                                                        ______________________________________                                                    Production                                                           amount of Yield                                                               L-lysine versus Color Accumulation                                            hydro- sugar of of                                                           Bacterial strain chloride (g/l) (%) broth SDAP                              ______________________________________                                        C/pCAB1     2.31      5.7     brown +                                           C/pCAB1 + pdapD 2.43 6.0 yellow +                                             C/pCAB1 + pdapE 2.35 5.8 brown -                                              C/pCAB1 + pdapF 2.23 5.5 brown +                                              C/pCABDE1 2.59 6.4 yellow -                                                   C/pCABDE1 + pdapF 2.43 6.0 yellow -                                           C/pCABD2 2.59 6.4 yellow -                                                  ______________________________________                                    

It was found that the two steps of dapD and dapE also concerned the ratedetermining in the C strain in the same manner as in the W3110 strain.

As described above, the strains of K-12 and C belonging to the differentseries had the same rate determining order. Thus it is believed that theentire species of E. coli can be applied with the concept that theL-lysine productivity can be improved in a stepwise manner by performingintroduction of dapA* and lysC* and enhancement of dapB and DDH (or dapDand dapE) in this order.

Industrial Applicability

According to the present invention, there has been obtained a DDPSmutant gene originating from a bacterium belonging to the genusEscherichia in which feedback inhibition by L-lysine is sufficientlydesensitized. An L-lysine-producing bacterium more improved than thosein the prior art has been able to be obtained by introducing the geneinto a bacterium belonging to the genus Escherichia harboring anaspartokinase in which feedback inhibition by L-lysine is desensitized.

Further, the L-lysine productivity can be improved in a stepwise mannerby enhancing dapB and DDH (or dapD and dapE) of the aforementionedL-lysine-producing bacterium in this order.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 20                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCGCAACTAC TGACATGACG            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - AGTAAGCCAT CAAATCTCCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1197 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: ESCHERICHIA - #COLI                                             (B) STRAIN: MC1061                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: prim.sub.-- - #transcript                                       (B) LOCATION: 248                                                             (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 272..1150                                                       (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: primer.sub.-- - #bind                                           (B) LOCATION: 27..46                                                          (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: primer.sub.-- - #bind                                           (B) LOCATION: 1156..1175                                                      (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: RBS                                                             (B) LOCATION: 261..265                                                        (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CCAGGCGACT GTCTTCAATA TTACAGCCGC AACTACTGAC ATGACGGGTG AT -             #GGTGTTCA     60                                                                 - - CAATTCCACG GCGATCGGCA CCCAACGCAG TGATCACCAG ATAATGTGTT GC -            #GATGACAG    120                                                                 - - TGTCAAACTG GTTATTCCTT TAAGGGGTGA GTTGTTCTTA AGGAAAGCAT AA -            #AAAAAACA    180                                                                 - - TGCATACAAC AATCAGAACG GTTCTGTCTG CTTGCTTTTA ATGCCATACC AA -            #ACGTACCA    240                                                                 - - TTGAGACACT TGTTTGCACA GAGGATGGCC C ATG TTC ACG GGA - #AGT ATT GTC           292                                                                                         - #                 Met - # Phe Thr Gly Ser Ile Val                           - #                  - # 1               5                   - - GCG ATT GTT ACT CCG ATG GAT GAA AAA GGT AA - #T GTC TGT CGG GCT AGC          340                                                                       Ala Ile Val Thr Pro Met Asp Glu Lys Gly As - #n Val Cys Arg Ala Ser                    10         - #         15         - #         20                      - - TTG AAA AAA CTG ATT GAT TAT CAT GTC GCC AG - #C GGT ACT TCG GCG ATC          388                                                                       Leu Lys Lys Leu Ile Asp Tyr His Val Ala Se - #r Gly Thr Ser Ala Ile                25             - #     30             - #     35                          - - GTT TCT GTT GGC ACC ACT GGC GAG TCC GCT AC - #C TTA AAT CAT GAC GAA          436                                                                       Val Ser Val Gly Thr Thr Gly Glu Ser Ala Th - #r Leu Asn His Asp Glu            40                 - # 45                 - # 50                 - # 55       - - CAT GCT GAT GTG GTG ATG ATG ACG CTG GAT CT - #G GCT GAT GGG CGC ATT          484                                                                       His Ala Asp Val Val Met Met Thr Leu Asp Le - #u Ala Asp Gly Arg Ile                            60 - #                 65 - #                 70              - - CCG GTA ATT GCC GGG ACC GGC GCT AAC GCT AC - #T GCG GAA GCC ATT AGC          532                                                                       Pro Val Ile Ala Gly Thr Gly Ala Asn Ala Th - #r Ala Glu Ala Ile Ser                        75     - #             80     - #             85                  - - CTG ACG CAG CGC TTC AAT GAC AGT GGT ATC GT - #C GGC TGC CTG ACG GTA          580                                                                       Leu Thr Gln Arg Phe Asn Asp Ser Gly Ile Va - #l Gly Cys Leu Thr Val                    90         - #         95         - #        100                      - - ACC CCT TAC TAC AAT CGT CCG TCG CAA GAA GG - #T TTG TAT CAG CAT TTC          628                                                                       Thr Pro Tyr Tyr Asn Arg Pro Ser Gln Glu Gl - #y Leu Tyr Gln His Phe               105              - #   110              - #   115                          - - AAA GCC ATC GCT GAG CAT ACT GAC CTG CCG CA - #A ATT CTG TAT AAT GTG          676                                                                       Lys Ala Ile Ala Glu His Thr Asp Leu Pro Gl - #n Ile Leu Tyr Asn Val           120                 1 - #25                 1 - #30                 1 -      #35                                                                              - - CCG TCC CGT ACT GGC TGC GAT CTG CTC CCG GA - #A ACG GTG GGC CGT        CTG      724                                                                    Pro Ser Arg Thr Gly Cys Asp Leu Leu Pro Gl - #u Thr Val Gly Arg Leu                          140  - #               145  - #               150              - - GCG AAA GTA AAA AAT ATT ATC GGA ATC AAA GA - #G GCA ACA GGG AAC TTA          772                                                                       Ala Lys Val Lys Asn Ile Ile Gly Ile Lys Gl - #u Ala Thr Gly Asn Leu                       155      - #           160      - #           165                  - - ACG CGT GTA AAC CAG ATC AAA GAG CTG GTT TC - #A GAT GAT TTT GTT CTG          820                                                                       Thr Arg Val Asn Gln Ile Lys Glu Leu Val Se - #r Asp Asp Phe Val Leu                   170          - #       175          - #       180                      - - CTG AGC GGC GAT GAT GCG AGC GCG CTG GAC TT - #C ATG CAA TTG GGC GGT          868                                                                       Leu Ser Gly Asp Asp Ala Ser Ala Leu Asp Ph - #e Met Gln Leu Gly Gly               185              - #   190              - #   195                          - - CAT GGG GTT ATT TCC GTT ACG ACT AAC GTC GC - #A GCG CGT GAT ATG GCC          916                                                                       His Gly Val Ile Ser Val Thr Thr Asn Val Al - #a Ala Arg Asp Met Ala           200                 2 - #05                 2 - #10                 2 -      #15                                                                              - - CAG ATG TGC AAA CTG GCA GCA GAA GAA CAT TT - #T GCC GAG GCA CGC        GTT      964                                                                    Gln Met Cys Lys Leu Ala Ala Glu Glu His Ph - #e Ala Glu Ala Arg Val                          220  - #               225  - #               230              - - ATT AAT CAG CGT CTG ATG CCA TTA CAC AAC AA - #A CTA TTT GTC GAA CCC         1012                                                                       Ile Asn Gln Arg Leu Met Pro Leu His Asn Ly - #s Leu Phe Val Glu Pro                       235      - #           240      - #           245                  - - AAT CCA ATC CCG GTG AAA TGG GCA TGT AAG GA - #A CTG GGT CTT GTG GCG         1060                                                                       Asn Pro Ile Pro Val Lys Trp Ala Cys Lys Gl - #u Leu Gly Leu Val Ala                   250          - #       255          - #       260                      - - ACC GAT ACG CTG CGC CTG CCA ATG ACA CCA AT - #C ACC GAC AGT GGT CGT         1108                                                                       Thr Asp Thr Leu Arg Leu Pro Met Thr Pro Il - #e Thr Asp Ser Gly Arg               265              - #   270              - #   275                          - - GAG ACG GTC AGA GCG GCG CTT AAG CAT GCC GG - #T TTG CTG TAA                 - #1150                                                                    Glu Thr Val Arg Ala Ala Leu Lys His Ala Gl - #y Leu Leu  *                    280                 2 - #85                 2 - #90                            - - AGTTTAGGGA GATTTGATGG CTTACTCTGT TCAAAAGTCG CGCCTGG   - #                  1197                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  292 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Phe Thr Gly Ser Ile Val Ala Ile Val Th - #r Pro Met Asp Glu Lys        1               5 - #                 10 - #                 15              - - Gly Asn Val Cys Arg Ala Ser Leu Lys Lys Le - #u Ile Asp Tyr His Val                   20     - #             25     - #             30                  - - Ala Ser Gly Thr Ser Ala Ile Val Ser Val Gl - #y Thr Thr Gly Glu Ser               35         - #         40         - #         45                      - - Ala Thr Leu Asn His Asp Glu His Ala Asp Va - #l Val Met Met Thr Leu           50             - #     55             - #     60                          - - Asp Leu Ala Asp Gly Arg Ile Pro Val Ile Al - #a Gly Thr Gly Ala Asn       65                 - # 70                 - # 75                 - # 80       - - Ala Thr Ala Glu Ala Ile Ser Leu Thr Gln Ar - #g Phe Asn Asp Ser Gly                       85 - #                 90 - #                 95              - - Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Ty - #r Asn Arg Pro Ser Gln                  100      - #           105      - #           110                  - - Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Al - #a Glu His Thr Asp Leu              115          - #       120          - #       125                      - - Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg Th - #r Gly Cys Asp Leu Leu          130              - #   135              - #   140                          - - Pro Glu Thr Val Gly Arg Leu Ala Lys Val Ly - #s Asn Ile Ile Gly Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Glu Ala Thr Gly Asn Leu Thr Arg Val As - #n Gln Ile Lys Glu        Leu                                                                                             165  - #               170  - #               175             - - Val Ser Asp Asp Phe Val Leu Leu Ser Gly As - #p Asp Ala Ser Ala Leu                  180      - #           185      - #           190                  - - Asp Phe Met Gln Leu Gly Gly His Gly Val Il - #e Ser Val Thr Thr Asn              195          - #       200          - #       205                      - - Val Ala Ala Arg Asp Met Ala Gln Met Cys Ly - #s Leu Ala Ala Glu Glu          210              - #   215              - #   220                          - - His Phe Ala Glu Ala Arg Val Ile Asn Gln Ar - #g Leu Met Pro Leu His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pr - #o Val Lys Trp Ala        Cys                                                                                             245  - #               250  - #               255             - - Lys Glu Leu Gly Leu Val Ala Thr Asp Thr Le - #u Arg Leu Pro Met Thr                  260      - #           265      - #           270                  - - Pro Ile Thr Asp Ser Gly Arg Glu Thr Val Ar - #g Ala Ala Leu Lys His              275          - #       280          - #       285                      - - Ala Gly Leu Leu                                                              290                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CTTCCCTTGT GCCAAGGCTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GAATTCCTTT GCGAGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2147 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: ESCHERICHIA - #COLI                                             (B) STRAIN: MC1061                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -35.sub.-- - #signal                                            (B) LOCATION: 242..249                                                        (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -10.sub.-- - #signal                                            (B) LOCATION: 265..273                                                        (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: primer.sub.-- - #bind                                           (B) LOCATION: 536..555                                                        (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: RBS                                                             (B) LOCATION: 575..578                                                        (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 584..1933                                                       (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: terminator                                                      (B) LOCATION: 1941..1968                                                      (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: S"          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: primer.sub.-- - #bind                                           (B) LOCATION: 2128..2147                                                      (D) OTHER INFORMATION: - #/note= "IDENTIFICATION METHOD: E"          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TCGAAGTGTT TCTGTAGTGC CTGCCAGGCA GCGGTCTGCG TTGGATTGAT GT -             #TTTTCATT     60                                                                 - - AGCAATACTC TTCTGATTTT GAGAATTGTG ACTTTGGAAG ATTGTAGCGC CA -            #GTCACAGA    120                                                                 - - AAAATGTGAT GGTTTTAGTG CCGTTAGCGT AATGTTGAGT GTAAACCCTT AG -            #CGCAGTGA    180                                                                 - - AGCATTTATT AGCTGAACTA CTGACCGCCA GGAGTGGATG AAAAATCCGC AT -            #GACCCCAT    240                                                                 - - CGTTGACAAC CGCCCCGCTC ACCCTTTATT TATAAATGTA CTACCTGCGC TA -            #GCGCAGGC    300                                                                 - - CAGAAGAGGC GCGTTGCCCA AGTAACGGTG TTGGAGGAGC CAGTCCTGTG AT -            #AACACCTG    360                                                                 - - AGGGGGTGCA TCGCCGAGGT GATTGAACGG CTGGCCACGT TCATCATCGG CT -            #AAGGGGGC    420                                                                 - - TGAATCCCCT GGGTTGTCAC CAGAAGCGTT CGCAGTCGGG CGTTTCGCAA GT -            #GGTGGAGC    480                                                                 - - ACTTCTGGGT GAAAATAGTA GCGAAGTATC GCTCTGCGCC CACCCGTCTT CC -            #GCTCTTCC    540                                                                 - - CTTGTGCCAA GGCTGAAAAT GGATCCCCTG ACACGAGGTA GTT ATG TC - #T GAA        ATT      595                                                                                      - #                  - #            Met Ser Glu Il -      #e                                                                                                - #                  - #              1                      - - GTT GTC TCC AAA TTT GGC GGT ACC AGC GTA GC - #T GAT TTT GAC GCC        ATG      643                                                                    Val Val Ser Lys Phe Gly Gly Thr Ser Val Al - #a Asp Phe Asp Ala Met            5                - #  10                - #  15                - #  20       - - AAC CGC AGC GCT GAT ATT GTG CTT TCT GAT GC - #C AAC GTG CGT TTA GTT          691                                                                       Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Al - #a Asn Val Arg Leu Val                            25 - #                 30 - #                 35              - - GTC CTC TCG GCT TCT GCT GGT ATC ACT AAT CT - #G CTG GTC GCT TTA GCT          739                                                                       Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Le - #u Leu Val Ala Leu Ala                        40     - #             45     - #             50                  - - GAA GGA CTG GAA CCT GGC GAG CGA TTC GAA AA - #A CTC GAC GCT ATC CGC          787                                                                       Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Ly - #s Leu Asp Ala Ile Arg                    55         - #         60         - #         65                      - - AAC ATC CAG TTT GCC ATT CTG GAA CGT CTG CG - #T TAC CCG AAC GTT ATC          835                                                                       Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Ar - #g Tyr Pro Asn Val Ile                70             - #     75             - #     80                          - - CGT GAA GAG ATT GAA CGT CTG CTG GAG AAC AT - #T ACT GTT CTG GCA GAA          883                                                                       Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Il - #e Thr Val Leu Ala Glu            85                 - # 90                 - # 95                 - #100       - - GCG GCG GCG CTG GCA ACG TCT CCG GCG CTG AC - #A GAT GAG CTG GTC AGC          931                                                                       Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Th - #r Asp Glu Leu Val Ser                           105  - #               110  - #               115              - - CAC GGC GAG CTG ATG TCG ACC CTG CTG TTT GT - #T GAG ATC CTG CGC GAA          979                                                                       His Gly Glu Leu Met Ser Thr Leu Leu Phe Va - #l Glu Ile Leu Arg Glu                       120      - #           125      - #           130                  - - CGC GAT GTT CAG GCA CAG TGG TTT GAT GTA CG - #T AAA GTG ATG CGT ACC         1027                                                                       Arg Asp Val Gln Ala Gln Trp Phe Asp Val Ar - #g Lys Val Met Arg Thr                   135          - #       140          - #       145                      - - AAC GAC CGA TTT GGT CGT GCA GAG CCA GAT AT - #A GCC GCG CTG GCG GAA         1075                                                                       Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Il - #e Ala Ala Leu Ala Glu               150              - #   155              - #   160                          - - CTG GCC GCG CTG CAG CTG CTC CCA CGT CTC AA - #T GAA GGC TTA GTG ATC         1123                                                                       Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu As - #n Glu Gly Leu Val Ile           165                 1 - #70                 1 - #75                 1 -      #80                                                                              - - ACC CAG GGA TTT ATC GGT AGC GAA AAT AAA GG - #T CGT ACA ACG ACG        CTT     1171                                                                    Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gl - #y Arg Thr Thr Thr Leu                          185  - #               190  - #               195              - - GGC CGT GGA GGC AGC GAT TAT ACG GCA GCC TT - #G CTG GCG GAG GCT TTA         1219                                                                       Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Le - #u Leu Ala Glu Ala Leu                       200      - #           205      - #           210                  - - CAC GCA TCT CGT GTT GAT ATC TGG ACC GAC GT - #C CCG GGC ATC TAC ACC         1267                                                                       His Ala Ser Arg Val Asp Ile Trp Thr Asp Va - #l Pro Gly Ile Tyr Thr                   215          - #       220          - #       225                      - - ACC GAT CCA CGC GTA GTT TCC GCA GCA AAA CG - #C ATT GAT GAA ATC GCG         1315                                                                       Thr Asp Pro Arg Val Val Ser Ala Ala Lys Ar - #g Ile Asp Glu Ile Ala               230              - #   235              - #   240                          - - TTT GCC GAA GCG GCA GAG ATG GCA ACT TTT GG - #T GCA AAA GTA CTG CAT         1363                                                                       Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gl - #y Ala Lys Val Leu His           245                 2 - #50                 2 - #55                 2 -      #60                                                                              - - CCG GCA ACG TTG CTA CCC GCA GTA CGC AGC GA - #T ATC CCG GTC TTT        GTC     1411                                                                    Pro Ala Thr Leu Leu Pro Ala Val Arg Ser As - #p Ile Pro Val Phe Val                          265  - #               270  - #               275              - - GGC TCC AGC AAA GAC CCA CGC GCA GGT GGT AC - #G CTG GTG TGC AAT AAA         1459                                                                       Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Th - #r Leu Val Cys Asn Lys                       280      - #           285      - #           290                  - - ACT GAA AAT CCG CCG CTG TTC CGC GCT CTG GC - #G CTT CGT CGC AAT CAG         1507                                                                       Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Al - #a Leu Arg Arg Asn Gln                   295          - #       300          - #       305                      - - ACT CTG CTC ACT TTG CAC AGC CTG AAT ATG CT - #G CAT TCT CGC GGT TTC         1555                                                                       Thr Leu Leu Thr Leu His Ser Leu Asn Met Le - #u His Ser Arg Gly Phe               310              - #   315              - #   320                          - - CTC GCG GAA GTT TTC GGC ATC CTC GCG CGG CA - #T AAT ATT TCG GTA GAC         1603                                                                       Leu Ala Glu Val Phe Gly Ile Leu Ala Arg Hi - #s Asn Ile Ser Val Asp           325                 3 - #30                 3 - #35                 3 -      #40                                                                              - - TTA ATC ACC ACG TCA GAA GTG AGC GTG GCA TT - #A ACC CTT GAT ACC        ACC     1651                                                                    Leu Ile Thr Thr Ser Glu Val Ser Val Ala Le - #u Thr Leu Asp Thr Thr                          345  - #               350  - #               355              - - GGT TCA ACC TCC ACT GGC GAT ACG TTG CTG AC - #G CAA TCT CTG CTG ATG         1699                                                                       Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Th - #r Gln Ser Leu Leu Met                       360      - #           365      - #           370                  - - GAG CTT TCC GCA CTG TGT CGG GTG GAG GTG GA - #A GAA GGT CTG GCG CTG         1747                                                                       Glu Leu Ser Ala Leu Cys Arg Val Glu Val Gl - #u Glu Gly Leu Ala Leu                   375          - #       380          - #       385                      - - GTC GCG TTG ATT GGC AAT GAC CTG TCA AAA GC - #C TGC GGC GTT GGC AAA         1795                                                                       Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Al - #a Cys Gly Val Gly Lys               390              - #   395              - #   400                          - - GAG GTA TTC GGC GTA CTG GAA CCG TTC AAC AT - #T CGC ATG ATT TGT TAT         1843                                                                       Glu Val Phe Gly Val Leu Glu Pro Phe Asn Il - #e Arg Met Ile Cys Tyr           405                 4 - #10                 4 - #15                 4 -      #20                                                                              - - GGC GCA TCC AGC CAT AAC CTG TGC TTC CTG GT - #G CCC GGC GAA GAT        GCC     1891                                                                    Gly Ala Ser Ser His Asn Leu Cys Phe Leu Va - #l Pro Gly Glu Asp Ala                          425  - #               430  - #               435              - - GAG CAG GTG GTG CAA AAA CTG CAT AGT AAT TT - #G TTT GAG TAA                 - #1933                                                                    Glu Gln Val Val Gln Lys Leu His Ser Asn Le - #u Phe Glu  *                                440      - #           445      - #           450                  - - ATACTGTATG GCCTGGAAGC TATATTTCGG GCCGTATTGA TTTTCTTGTC AC -             #TATGCTCA   1993                                                                 - - TCAATAAACG AGCCTGTACT CTGTTAACCA GCGTCTTTAT CGGAGAATAA TT -            #GCCTTTAA   2053                                                                 - - TTTTTTTATC TGCATCTCTA ATTAATTATC GAAAGAGATA AATAGTTAAG AG -            #AAGGCAAA   2113                                                                 - - ATGAATATTA TCAGTTCTGC TCGCAAAGGA ATTC       - #                  -     #      2147                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  449 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Ser Glu Ile Val Val Ser Lys Phe Gly Gl - #y Thr Ser Val Ala Asp        1               5 - #                 10 - #                 15              - - Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Va - #l Leu Ser Asp Ala Asn                   20     - #             25     - #             30                  - - Val Arg Leu Val Val Leu Ser Ala Ser Ala Gl - #y Ile Thr Asn Leu Leu               35         - #         40         - #         45                      - - Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Gl - #u Arg Phe Glu Lys Leu           50             - #     55             - #     60                          - - Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Le - #u Glu Arg Leu Arg Tyr       65                 - # 70                 - # 75                 - # 80       - - Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Le - #u Leu Glu Asn Ile Thr                       85 - #                 90 - #                 95              - - Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Se - #r Pro Ala Leu Thr Asp                  100      - #           105      - #           110                  - - Glu Leu Val Ser His Gly Glu Leu Met Ser Th - #r Leu Leu Phe Val Glu              115          - #       120          - #       125                      - - Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Tr - #p Phe Asp Val Arg Lys          130              - #   135              - #   140                          - - Val Met Arg Thr Asn Asp Arg Phe Gly Arg Al - #a Glu Pro Asp Ile Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Le - #u Pro Arg Leu Asn        Glu                                                                                             165  - #               170  - #               175             - - Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Se - #r Glu Asn Lys Gly Arg                  180      - #           185      - #           190                  - - Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Ty - #r Thr Ala Ala Leu Leu              195          - #       200          - #       205                      - - Ala Glu Ala Leu His Ala Ser Arg Val Asp Il - #e Trp Thr Asp Val Pro          210              - #   215              - #   220                          - - Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Se - #r Ala Ala Lys Arg Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Me - #t Ala Thr Phe Gly        Ala                                                                                             245  - #               250  - #               255             - - Lys Val Leu His Pro Ala Thr Leu Leu Pro Al - #a Val Arg Ser Asp Ile                  260      - #           265      - #           270                  - - Pro Val Phe Val Gly Ser Ser Lys Asp Pro Ar - #g Ala Gly Gly Thr Leu              275          - #       280          - #       285                      - - Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Ph - #e Arg Ala Leu Ala Leu          290              - #   295              - #   300                          - - Arg Arg Asn Gln Thr Leu Leu Thr Leu His Se - #r Leu Asn Met Leu His      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Il - #e Leu Ala Arg His        Asn                                                                                             325  - #               330  - #               335             - - Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Va - #l Ser Val Ala Leu Thr                  340      - #           345      - #           350                  - - Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly As - #p Thr Leu Leu Thr Gln              355          - #       360          - #       365                      - - Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Ar - #g Val Glu Val Glu Glu          370              - #   375              - #   380                          - - Gly Leu Ala Leu Val Ala Leu Ile Gly Asn As - #p Leu Ser Lys Ala Cys      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Val Gly Lys Glu Val Phe Gly Val Leu Gl - #u Pro Phe Asn Ile        Arg                                                                                             405  - #               410  - #               415             - - Met Ile Cys Tyr Gly Ala Ser Ser His Asn Le - #u Cys Phe Leu Val Pro                  420      - #           425      - #           430                  - - Gly Glu Asp Ala Glu Gln Val Val Gln Lys Le - #u His Ser Asn Leu Phe              435          - #       440          - #       445                      - - Glu                                                                          450                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CTTTCACTGA TATCCCTCCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AAAAAGTGGA CCAAATGGTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CATCTAAGTA TGCATCTCGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - TGCCCCTCGA GCTAAATTAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TGCACGGTAG GATGTAATCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TTAATGAAAC AAATGCCCGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TTTATTCATA ATTGCCACCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CACGGTAATA CATATAACCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CCTGCAATTG TCAAACGTCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GTCGACGCGC TTGAGATCTT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TCATAAAGAG TCGCTAAACG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "SYNTHETIC DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CAACCGCCCG GTCATCAAGC            - #                  - #                      - # 20                                                                 __________________________________________________________________________

What is claimed is:
 1. An isolated DNA coding for a dihydrodipicolinatesynthase originating from a bacterium belonging to the genusEscherichia, wherein the dihydrodipicolinate synthase has a mutationwhich desensitizes feedback inhibition by L-lysine, wherein the mutationis selected from the group consisting of(a) a mutation to replace thealanine residue at the 81st position as counted from the N-terminal inthe amino acid sequence of the dihydrodipicolinate synthase of SEQ IDNO: 4 with another amino acid residue, (b) a mutation to replace thehistidine residue at the 118th position as counted from the N-terminalin the amino acid sequence of the dihydrodipicolinate synthase of SEQ IDNO: 4 with another amino acid residue, and (c) a mutation to replace thealanine residue at the 81st position as counted from the N-terminal inthe amino acid sequence of the dihydrodipicolinate synthase of SEQ IDNO: 4 with another amino acid residue and replace the histidine residueat the 118th position as counted from the N-terminal in the amino acidsequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 withanother amino acid residue, (d) a mutation to replace the alanineresidue corresponding to the 81st position as counted from theN-terminal in the amino acid sequence of the dihydrodipicolinatesynthase of SEQ ID NO: 4 with another amino acid residue, (e) a mutationto replace the histidine residue corresponding to the 118th position ascounted from the N-terminal in the amino acid sequence of thedihydrodipicolinate synthase of SEQ ID NO: 4 with another amino acidresidue, and (f) a mutation to replace the alanine residue correspondingto the 81st position as counted from the N-terminal in the amino acidsequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 withanother amino acid residue and replace the histidine residuecorresponding to the 118th position as counted from the N-terminal inthe amino acid sequence of the dihydrodipicolinate synthase of SEQ IDNO: 4 with another amino acid residue.
 2. The isolated DNA of claim 1,wherein the mutation to desensitize feedback inhibition by L-lysine isselected from the group consisting of(a) a mutation to replace thealanine residue at the 81st position as counted from the N-terminal inthe amino acid sequence of the dihydrodipicolinate synthase of SEQ IDNO: 4 with a valine residue, (b) a mutation to replace the histidineresidue at the 118th position as counted from the N-terminal in theamino acid sequence of the dihydrodipicolinate synthase of SEQ ID NO: 4with a tyrosine residue, and (c) a mutation to replace the alanineresidue at the 81^(st) position as counted from the N-terminal in theamino acid sequence of the dihydrodipicolinate synthase of SEQ ID NO: 4with a valine residue and replace the 118th histidine residue as countedfrom the N-terminal in the amino acid sequence of thedihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosine residue,(d) a mutation to replace the alanine residue corresponding to the 81stposition as counted from the N-terminal in the amino acid sequence ofthe dihydrodipicolinate synthase of SEQ ID NO: 4 with a valine residue,(e) a mutation to replace the histidine residue corresponding to the118th position as counted from the N-terminal in the amino acid sequenceof the dihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosineresidue, and (f) a mutation to replace the alanine residue correspondingto the 81^(st) position as counted from the N-terminal in the amino acidsequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 with avaline residue and replace the histidine residue corresponding to the118th residue as counted from the N-terminal in the amino acid sequenceof the dihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosineresidue.
 3. A bacterium belonging the genus Escherichia which istransformed with a DNA coding for a dihydrodipicolinate synthaseoriginating from a bacterium belonging to the genus Escherichia andhaving mutation to desensitize feedback inhibition by L-lysine, whereinthe mutation is selected from the group consisting of(a) a mutation toreplace the alanine residue at the 81st position as counted from theN-terminal in the amino acid sequence of the dihydrodipicolinatesynthase of SEQ ID NO: 4 with another amino acid residue, (b) a mutationto replace the histidine residue at the 118th position as counted fromthe N-terminal in the amino acid sequence of the dihydrodipicolinatesynthase of SEQ ID NO: 4 with another amino acid residue, and (c) amutation to replace the alanine residue at the 81st position as countedfrom the N-terminal in the amino acid sequence of thedihydrodipicolinate synthase of SEQ ID NO: 4 with another amino acidresidue and replace the histidine residue at the 118th position ascounted from the N-terminal in the amino acid sequence of thedihydrodipicolinate synthase of SEQ ID NO: 4 with another amino acidresidue, (d) a mutation to replace the alanine residue corresponding tothe 81st position as counted from the N-terminal in the amino acidsequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 withanother amino acid residue, (e) a mutation to replace the histidineresidue corresponding to the 118th position as counted from theN-terminal in the amino acid sequence of the dihydrodipicolinatesynthase of SEQ ID NO: 4 with another amino acid residue, and (f) amutation to replace the alanine residue corresponding to the 81stposition as counted from the N-terminal in the amino acid sequence ofthe dihydrodipicolinate synthase of SEQ ID NO: 4 with another amino acidresidue and replace the histidine residue corresponding to the 118thposition as counted from the N-terminal in the amino acid sequence ofthe dihydrodipicolinate synthase of SEQ ID NO: 4 with another amino acidresidue.
 4. The bacterium of claim 3, wherein the mutation is selectedfrom the group consisting of(a) a mutation to replace the alanineresidue at the 81st position as counted from the N-terminal in the aminoacid sequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 with avaline residue, (b) a mutation to replace the histidine residue at the118th position as counted from the N-terminal in the amino acid sequenceof the dihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosineresidue, and (c) a mutation to replace the alanine residue at the 81stposition as counted from the N-terminal in the amino acid sequence ofthe dihydrodipicolinate synthase of SEQ ID NO: 4 with a valine residueand replace the histidine residue at the 118th position as counted fromthe N-terminal in the amino acid sequence of the dihydrodipicolinatesynthase of SEQ ID NO: 4 with a tyrosine residue, (d) a mutation toreplace the alanine residue corresponding to the 81st position ascounted from the N-terminal in the amino acid sequence of thedihydrodipicolinate synthase of SEQ ID NO: 4 with a valine residue, (e)a mutation to replace the histidine residue corresponding to the 118thposition as counted from the N-terminal in the amino acid sequence ofthe dihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosineresidue, and (f) a mutation to replace the alanine residue correspondingto the 81st position as counted from the N-terminal in the amino acidsequence of the dihydrodipicolinate synthase of SEQ ID NO: 4 with avaline residue and replace the histidine residue corresponding to the118th position as counted from the N-terminal in the amino acid sequenceof the dihydrodipicolinate synthase of SEQ ID NO: 4 with a tyrosineresidue.
 5. The bacterium of claim 3, further harboring an aspartokinasewhich is desensitized to feedback inhibition by L-lysine.
 6. Thebacterium of claim 5, which is obtained by introducing, into its cells,a DNA coding for an aspartokinase III originating from a bacteriumbelonging to the genus Escherichia, wherein the aspartokinase III has amutation which desensitizes feedback inhibition by L-lysine.
 7. Thebacterium of claim 6, wherein the mutation is selected from the groupconsisting of(a) a mutation to replace the glycine residue at the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(b) a mutation to replace the glycine residue at the 323rd position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO:8 with another amino acid residue andreplace the glycine residue at the 408th position as counted from theN-terminal in the amino acid sequence of the aspartokinase III of SEQ IDNO: 8 with another amino acid residue, (c) a mutation to replace theglycine residue at the 34th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 withanother amino acid residue and replace the glycine residue at the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(d) a mutation to replace the leucine residue at the 325th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with another amino acid residue, (e) amutation to replace the methionine residue at the 318th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with another amino acid residue, (f) amutation to replace the methionine residue at the 318th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with another amino acid residue andreplace the valine residue at the 349th position as counted from theN-terminal in the amino acid sequence of the aspartokinase III of SEQ IDNO: 8 with another amino acid residue, (g) a mutation to replace theserine residue at the 345th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 withanother amino acid residue, (h) a mutation to replace the valine residueat the 347th position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with another aminoacid residue, (i) a mutation to replace the threonine residue at the352nd position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with another amino acidresidue, (j) a mutation to replace the threonine residue at the 352ndposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residueand replace the serine residue at the 369th position as counted from theN-terminal in the amino acid sequence of the aspartokinase III of SEQ IDNO: 8 with another amino acid residue, (k) a mutation to replace theglutamic acid residue at the 164th position as counted from theN-terminal in the amino acid sequence of the aspartokinase III of SEQ IDNO: 8 with another amino acid residue, and (l) a mutation to replace themethionine residue at the 417th position as counted from the N-terminalin the amino acid sequence of the aspartokinase III of SEQ ID NO: 8 withanother amino acid residue and replace the cysteine residue at the 419thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(m) a mutation to replace the glycine residue corresponding to the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(n) a mutation to replace the glycine residue corresponding to the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO:8 with another amino acid residue andreplace the glycine residue corresponding to the 408th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with another amino acid residue, (o) amutation to replace the glycine residue corresponding to the 34thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO:8 with another amino acid residue andreplace the glycine residue corresponding to the 323rd position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with another amino acid residue, (p) amutation to replace the leucine residue corresponding to the 325thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(q) a mutation to replace the methionine residue corresponding to the318th position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with another amino acidresidue, (r) a mutation to replace the methionine residue correspondingto the 318th position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with another aminoacid residue and replace the valine residue corresponding to the 349thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(s) a mutation to replace the serine residue corresponding to the 345thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(t) a mutation to replace the valine residue corresponding to the 347thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(u) a mutation to replace the threonine residue corresponding to the352nd position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with another amino acidresidue, (v) a mutation to replace the threonine residue correspondingto the 352nd position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with another aminoacid residue and replace the serine residue corresponding to the 369thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with another amino acid residue,(w) a mutation to replace the glutamic acid residue corresponding to the164th position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with another amino acidresidue, and (x) a mutation to replace the methionine residuecorresponding to the 417th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 withanother amino acid residue and replace the cysteine residuecorresponding to the 419th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 withanother amino acid residue.
 8. The bacterium of claim 7, wherein themutation is selected from the group consisting of(a) a mutation toreplace the glycine residue at the 323rd position as counted from theN-terminal in the amino acid sequence of the aspartokinase III of SEQ IDNO: 8 with an aspartic acid residue, (b) a mutation to replace theglycine residue at the 323rd position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with anaspartic acid residue and replace the glycine residue at the 408thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with an aspartic acid residue, (c)a mutation to replace the arginine residue at the 34th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO:8 with a cysteine residue and replace theglycine residue at the 323rd position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with anaspartic acid residue, (d) a mutation to replace the leucine residue atthe 325th position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with a phenylalanineresidue, (e) a mutation to replace the methionine residue at the 318thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with an isoleucine residue, (f) amutation to replace the methionine residue at the 318th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with an isoleucine residue and replacethe valine residue at the 349th position as counted from the N-terminalin the amino acid sequence of the aspartokinase III of SEQ ID NO: 8 witha methionine residue, (g) a mutation to replace the serine residue atthe 345th position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with a leucineresidue, (h) a mutation to replace the valine residue at the 347thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with a valine residue, (i) amutation to replace the threonine residue at the 352nd position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with an isoleucine residue, (j) amutation to replace the threonine residue at the 352nd position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with an isoleucine residue and replacethe serine residue at the 369th position as counted from the N-terminalin the amino acid sequence of the aspartokinase III of SEQ ID NO: 8 witha phenylalanine residue, (k) a mutation to replace the glutamic acidresidue at the 164th position as counted from the N-terminal in theamino acid sequence of the aspartokinase III of SEQ ID NO: 8 with alysine residue, and (l) a mutation to replace the methionine residue atthe 417th position as counted from the N-terminal in the amino acidsequence of the aspartokinase III of SEQ ID NO: 8 with an isoleucineresidue and replace the cysteine residue at the 419th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with a tyrosine residue, (m) amutation to replace the glycine residue corresponding to the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with an aspartic acid residue, (n)a mutation to replace the glycine residue corresponding to the 323rdposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with an aspartic acid residue andreplace the glycine residue corresponding to the 408th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with an aspartic acid residue, (o) amutation to replace the arginine residue corresponding to the 34thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO:8 with a cysteine residue and replacethe glycine residue corresponding to the 323rd position as counted fromthe N-terminal in the amino acid sequence of the aspartokinase III ofSEQ ID NO: 8 with an aspartic acid residue, (p) a mutation to replacethe leucine residue corresponding to the 325th position as counted fromthe N-terminal in the amino acid sequence of the aspartokinase III ofSEQ ID NO: 8 with a phenylalanine residue, (q) a mutation to replace themethionine residue corresponding to the 318th position as counted fromthe N-terminal in the amino acid sequence of the aspartokinase III ofSEQ ID NO: 8 with an isoleucine residue, (r) a mutation to replace themethionine residue corresponding to the 318th position as counted fromthe N-terminal in the amino acid sequence of the aspartokinase III ofSEQ ID NO: 8 with an isoleucine residue and replace the valine residuecorresponding to the 349th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with amethionine residue, (s) a mutation to replace the serine residuecorresponding to the 345th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with aleucine residue, (t) a mutation to replace the valine residuecorresponding to the 347th position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with avaline residue, (u) a mutation to replace the threonine residuecorresponding to the 352nd position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with anisoleucine residue, (v) a mutation to replace the threonine residuecorresponding to the 352nd position as counted from the N-terminal inthe amino acid sequence of the aspartokinase III of SEQ ID NO: 8 with anisoleucine residue and replace the serine residue corresponding to the369th position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with a phenylalanine residue,(w) a mutation to replace the glutamic acid residue corresponding to the164th position as counted from the N-terminal in the amino acid sequenceof the aspartokinase III of SEQ ID NO: 8 with a lysine residue, and (x)a mutation to replace the methionine residue corresponding to the 417thposition as counted from the N-terminal in the amino acid sequence ofthe aspartokinase III of SEQ ID NO: 8 with an isoleucine residue andreplace the cysteine residue corresponding to the 419th position ascounted from the N-terminal in the amino acid sequence of theaspartokinase III of SEQ ID NO: 8 with a tyrosine residue.
 9. Thebacterium of claim 5, wherein a dihydrodipicolinate reductase gene isenhanced.
 10. The bacterium of claim 9, transformed with a recombinantDNA constructed by ligating the dihydrodipicolinate reductase gene witha vector autonomously replicable in cells of bacteria belonging to thegenus Escherichia.
 11. The bacterium of claim 9, into which an enhanceddiaminopimelate dehydrogenase gene originating from coryneform bacteriumhas been introduced.
 12. The of claim 11, transformed with a recombinantDNA constructed by ligating the diaminopimelate dehydrogenase geneoriginating from a coryneform bacterium with a vector autonomouslyreplicable in cells of bacteria belonging to the genus Escherichia. 13.The bacterium of claim 9, wherein a succinyldiaminopimelate transaminasegene and a succinyldiaminopimelate transaminase gene and asuccinyldiaminopimelate deacylase gene are enhanced.
 14. The bacteriumof claim 13, transformed with a single recombinant DNA or tworecombinant DNA's constructed by ligating the succinyldiaminopimelatetransaminase gene and the succinyldiaminopimelate deacylase gene with anidentical vector or different vectors autonomously replicable in cellsof bacteria belonging to the genus Escherichia.
 15. A method ofproducing L-lysine, comprising:cultivating the bacterium of claim 3 in asuitable culture medium, producing and accumulating L-lysine in theculture thereof, and collecting L-lysine from the culture.
 16. Abacterium belonging the genus Escherichia which is transformed with aDNA coding for a dihydrodipicolinate synthase originating from abacterium belonging to the genus Escherichia and having mutation todesensitize feedback inhibition by L-lysine, andfurther harboring anaspartokinase which is desensitized to feedback inhibition by L-lysine,and wherein a dihydrodipicolinate reductase gene is enhanced.
 17. Thebacterium of claim 16, transformed with a recombinant DNA constructed byligating the dihydrodipicolinate reductase gene with a vectorautonomously replicable in cells of bacteria belonging to the genusEscherichia.
 18. The bacterium of claim 16, into which an enhanceddiaminopimelate dehydrogenase gene originating from coryneform bacteriumhas been introduced.
 19. The of claim 18, transformed with a recombinantDNA constructed by ligating the diaminopimelate dehydrogenase geneoriginating from a coryneform bacterium with a vector autonomouslyreplicable in cells of bacteria belonging to the genus Escherichia. 20.The bacterium of claim 16, wherein a succinyldiaminopimelatetransaminase gene and a succinyldiaminopimelate transaminase gene and asuccinyldiaminopimelate deacylase gene are enhanced.
 21. The bacteriumof claim 20, transformed with a single recombinant DNA or tworecombinant DNA's constructed by ligating the succinyldiaminopimelatetransaminase gene and the succinyldiaminopimelate deacylase gene with anidentical vector or different vectors autonomously replicable in cellsof bacteria belonging to the genus Escherichia.
 22. A method ofproducing L-lysine, comprising:cultivating the bacterium of claim 16 ina suitable culture medium, producing and accumulating L-lysine in theculture thereof, and collecting L-lysine from the culture.